T1 receptor-like ligand II and uses thereof

ABSTRACT

The present invention relates to a novel T1 Receptor (T1R)-like ligand II protein. In particular, isolated nucleic acid molecules are provided encoding the T1R-like ligand II protein. T1R-like ligand II polypeptides are also provided, as are recombinant vectors and host cells for expressing the same. This invention further relates to pharmaceutical compositions and formulations comprising T1R-like ligand II. Also provided are methods of using T1R-like ligand II polynucleotides, polypeptides, antibodies or agonists/antagonists for therapeutic and diagnostic purposes. Diagnostic kits are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of, and claims priority under 35 U.S.C.§120, to U.S. patent application Ser. No. 09/731,924, filed Dec. 8,2000, now U.S. Pat. No. 6,605,271, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application Ser. No. 60/169,979,filed Dec. 10, 1999; said U.S. patent application Ser. No. 09/731,924 isalso a Continuation-in-Part of, and claims priority under 35 U.S.C.§120, to U.S. patent application Ser. No. 09/317,641, filed May 25,1999, now U.S. Pat. No. 6,667,032, which is a Divisional of, and claimspriority under 35 U.S.C. §120, to U.S. patent application Ser. No.08/916,442, filed Aug. 22,1997, now U.S. Pat. No. 6,586,210, whichclaims priority under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication Ser. No. 60/024,348, filed Aug. 23, 1996; all of which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a novel T1 Receptor (T1R)-like ligandII protein. In particular, isolated nucleic acid molecules are providedencoding the T1R-like ligand II protein. T1R-like ligand II polypeptidesare also provided, as are recombinant vectors and host cells forexpressing the same. This invention further relates to pharmaceuticalcompositions and formulations comprising T1R-like ligand II. Alsoprovided are methods of using T1R-like ligand II polynucleotides,polypeptides, antibodies or agonists/antagonists for therapeutic anddiagnostic purposes. Diagnostic kits are further provided.

BACKGROUND OF THE INVENTION

Interleukin-1 (IL-1). Interleukin-1 (IL-1α and IL-1β) is a“multifunctional” cytokine that a effects n early every cell type, andoften in concert with other cytokines or small mediator molecules.(Dinarello, C. A., Blood 87:2095–2147 (Mar. 15, 1996).) There are threemembers of the IL-1 gene family: IL-1α, IL-1β and IL-1 receptorantagonist (IL-1Ra). IL-1α and IL-1β are agonists and IL-1Ra is aspecific receptor antagonist. IL-1α and β are synthesized as precursorswithout leader sequences. The molecular weight of each precursor is 31kD. Processing of IL-1α or IL-1β to “mature ” forms of 17 kD requiresspecific cellular proteases. In contrast, IL-1Ra evolved with a signalpeptide and is readily transported out of the cells and termed secretedIL-1Ra (sIL-1Ra).

IL-1 Receptor and Ligands. The receptors and ligands of the IL-1 pathwayhave been well defined (for review, see Dinarello, C. A., FASEB J.8:1314–1325 (1994); Sims, J. E. et al., Interleukin-1 signaltransduction: Advances in Cell and Molecular Biology of Membranes andOrganelles, Vol. 3, JAI Press, Inc., Greenwich, Conn. (1994), pp.197–222). Three ligands, IL-1α, IL-1β, and IL-1 receptor antagonist(IL-1Ra) bind three forms of IL-1 receptor, an 80-kDa type I IL-1receptor (IL-IR1) (Sims, J. E. et al., Science 241:585–589 (1988)),a68-kDa type II IL-1 receptor(IL-1RII) (McMahan, C. J. et al., EMBO J.10:2821–2832 (1991)), and a soluble form of the type II IL-1R (sIL-1RII)(Colotta, F. et al., Science 261:472–475 (1993)).

The interactions between the IL-1 ligands and receptors play anessential role in the stimulation and regulation of the IL-1-mediatedhost response to injury and infection. Cells expressing IL-1RI andtreated with IL-1α or IL-1β respond in several specific ways, includingstimulating nuclear localization of the rel-related transcriptionfactor, NF-κβ (for review, see Thanos, D. & Maniatis, T., Cell80:529–532 (1996)), activation of protein kinases of themitogen-activated protein kinase superfamily that phosphorylate residuethreonine 669 (Thr-669) of the epidermal growth factor receptor (EGFR)(Guy, G. R. et al., J. Biol. Chem. 267:1846–1852(1992); Bird, T. A. etal., J. Biol. Chem. 268:22861–22870 (1991); Bird, T. A. et al., J. Biol.chem. 269:31836–31844 (1994)), and stimulation of transcription oftheIL-8 gene (Mukaida, N. et al., J. Biol. chem. 265:21128–21133 (1990)).

IL-1RI-like family. Many proteins from diverse systems show homology tothe cytoplasmic domain ofthe IL-1RI. This expanding IL-1RI-like familyincludes mammalian proteins, Drosophila proteins, and a plant (tobacco)protein. (Gay, N. J. & Keith, F. J., Nature 351:355–356 (1991);Hashimoto, C. et al., Cell 52:269–279 (1988); Schneider, D. S. et al.,Genes & Dev. 5:797–807 (1991); Edon, E. et al., Development 120:885–899(1994); Mitchan, J. L. et al., J. Biol. Chem 271:5777–5782 (Mar. 8,1996)).

The mammalian IL-1RI-like receptor family members include a murineprotein MyD88 (Lord, K. A. et al., Oncogene 5:1095–1097 (1990)) and ahuman gene, rsc786 (Nomura,N. et al., DNA Res. 1:27–35(1994)). Anothermurine receptor member, T1/ST2, was previously characterized as a novelprimary response gene expressed in BALB/c-3T3 cells (Klemenz, R. et al.,Proc. Natl. Acad. Sci. USA 86:5708–5712 (1989); Tominaga, S., FEBS Lett.258:301–304 (1989); Tominga, S. et al., FEBS Lett. 318:83–87 (1993)).The transmembrane protein mulL-1R AcP (Greenfeder, S. A. et al., J.Biol. Chem. 270:13757–13765 (1995)) has homology to both the type I andtype II IL-1R. IL-1R AcP has recently been shown to increase theaffinity of IL-1RI for IL-1 and may be involved in mediating the IL-1response.

T1Receptors. T1/ST2receptors (hereinafter, “T1 receptors”), as a memberof the IL-1 receptor family (Bergers, G., et al., EMBO J. 13:1176(1994)), have various homologs in different species. In the rat, it iscalled Fit-1, an estrogen-inducible, c-fos-dependent transmembraneprotein that shares 26% to 29% amino acid homology to the mouse IL-1RIand II, respectively. In the mouse, the Fit-1 protein is called ST2 andin the human it is called T1. The organization of the two IL-1 receptorsand the Fit-1/ST2/T1 genes indicates they are derived from a commonancestor (Sims, J. E., et al., Cytokine 7:483 (1995)). Fit-1 exists intwo forms: a membrane form (Fit-1M) with a cytosolic domain similarly tothat of the IL-1 RI and Fit-1S, which is secreted and composed of theextracellular domain of Fit-M.

In many ways, these two forms of the Fit-1 protein are similar to thoseof the membrane-bound and soluble IL-1RI. It has been shown that theIL-1sRI is derived from proteolytic cleavage of the cell-bound form(Sims, J. E., et al., Cytokine 7:483 (1995)). On the other hand, theFit-1 gene is under the control of two promoters, which results in twoisoforms coding for either the membrane or soluble form of the receptor.Two RNA transcripts result from alternative RNA splicing of the 3′ endof the gene. Although IL-1β binds weakly to Fit-1 and does not transducea signal (Reikerstorger, A., et al., J. Biol. Chem. 270:17645 (1995)), achimeric receptor consisting ofthe extracellular murine IL-1RI fused tothe cytosolic Fit-1 transduces an IL-1 signal (Reikerstorger, A., etal., J. Biol. Chem. 270:17645 (1995)). The cytosolic portion of Fit-Ialign with GTPase-like sequences of IL-1RI (Hopp, T. P., Protein Sci.4:1851 (1995)) (see below).

IL-1 production in various disease states. Increased IL-1 production hasbeen reported in patients with various viral, bacterial, fungal, andparasitic infections; intravascular coagulation; high-dose IL-2 therapy;solid tumors; leukemias; Alzheimer's disease; HIV-1 infection;autoimmune disorders; trauma (surgery); hemodialysis; ischemic diseases(myocardial infarction); noninfectious hepatitis; asthma; UV radiation;closed head injury; pancreatitis; periodontitis; graft-versus-hostdisease; transplant rejection; and in healthy subjects after strenuousexercise. There is an association of increased IL-1β production inpatients with Alzheimer's disease and a possible role for IL-1 in therelease of the amyloid precursor protein (Vasilakos, J. P., et al., FEBSLett. 354:289 (1994)). However, in most conditions, IL-1 is not the onlycytokine exhibiting increased production and hence the specificity ofthe IL-1 findings as related to the pathogenesis of any particulardisease is lacking. In various disease states, IL-1β, but not IL-1α, isdetected in the circulation.

IL-1 in Therapy. Although IL-1 has been found to exhibit many importantbiological activities, it is also found to be toxic at doses that areclose to therapeutic dosages (Dinarello, C. A., Blood 87:2095–2147 (Mar.15, 1996)). In general, the acute toxicities of either isoform of IL-1were greater after intravenous compared with subcutaneous injection.Subcutaneous injection was associated with significant local pain,erythema, and swelling (Kitamura, T., & Takaku, F., Exp. Med. 7:170(1989); Laughlin, M. J., Ann. Hematol. 67:267 (1993)). Patientsreceiving intravenous IL-1 at doses of 100 ng/kg or greater experiencedsignificant hypotension. In patients receiving IL-1β from 4 to 32 ng/kgsubcutaneously, there was only one episode of hypotension at the highestdose level (Laughlin, M. J., Ann. Hematol. 67:267 (1993)).

Contrary to IL-1-associated myelostimulation in patients with normalmarrow reserves, patients with a plastic anemia treated with 5 dailydoses of IL-1α (30 to 100 ng/kg) had no increases in peripheral bloodcounts or bone marrow cellularity (Walsh, C. E., et al., Br. J. Haematol80:106 (1992)). IL-1 has been administered to patients undergoingvarious regiments of chemotherapy to reduce the nadir of neutropenia andthrombocytopenia.

Daily treatment with 40 ng/kg IL-1α from day 0 to day 13 of autologousbone marrow or stem cells resulted in an earlier recovery of neutropenia(median, 12 days; P<0.001) (Weisdorf, D., et al., Blood 84:2044 (1994)).After 14 days of treatment, the bone marrow was significantly enrichedwith committed myeloid progenitor cells. Similar results were reportedin patients with AML receiving 50 ng/kg/d of IL-1β for 5 days startingat the time of transplantation with purged or nonpurged bone marrow(Nemunaitis, J., et al., Blood 83:3473 (1994)). Injecting humans withlow doses of either IL-1α or IL-1β confirms the impressive pyrogenic andhypotension-inducing properties of the molecules.

Amelioration of Disease Using Soluble IL-1 Receptors. Administration ofmurine IL-1sRI to mice has increased the survival of heterotopic heartallografts and reduced the hyperplastic lymph node response toallogeneic cells (Fanslow, W. C., et al., Science 248:739 (1990)). In arat model of antigen-induced arthritis, local instillation of the murineIL-1sRI reduced joint swelling and tissue destruction (Dower, S. K., etal., Therapeutic Immunol. 1:113 (1994)). These data suggest that theamount of IL-1sRI administered in the normal, contralateral joint wasacting systemically. In a model of experimental autoimmuneencephalitits, the IL-1sRI reduced the severity of this disease (Jacobs,C. A., et al., J. Immunol. 146:2983 (1991)).

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a human T1 receptor-(T1R-)likeligand II polypeptide having the amino acid sequence in FIGS. 1A–B (SEQID NO:2). The T1R-like ligand II contains an open reading frame encodinga polypeptide of about 229 amino acid residues including an N-terminalmethionine, a leader sequence of about 26 amino acid residues, anextracellular mature domain of about 168 residues, a transmembranedomain of about 23 residues and an intracellular domain of about 12amino acid residues, and a deduced molecular weight of about 26 kDa. The203 amino acid sequence of the expected mature T1R-like ligand IIprotein is shown in SEQ ID NO:2 (amino acid residues 1–203).

The invention also provides isolated nucleic acid molecules encoding anT1R-like ligand II having an amino acid sequence encoded by the cDNA ofthe clone deposited as ATCC Deposit No. 97655 on Jul. 12, 1996.Preferably, the nucleic acid molecule will encode the mature polypeptideencoded by the above-described deposited cDNA.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the T1R-like ligand II polypeptide having the complete aminoacid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding theT1R-like ligand II polypeptide having the complete amino acid sequencein SEQ ID NO:2 but minus the N-terminal methionine residue; (c) anucleotide sequence encoding the mature T1R-like ligand II polypeptidehaving the amino acid sequence at positions from about 1 to about 203 inSEQ ID NO:2; (d) a nucleotide sequence encoding the T1R-like ligand IIpolypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97655; (e) a nucleotide sequenceencoding the mature T1R-like ligand II polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97655;and (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), or (f), above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), (d), (e),or (f), above. This polynucleotide which hybridizes does not hybridizeunder stringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a T1R-likeligand II polypeptide having an amino acid sequence in (a), (b), (c),(d), or (e), above.

The present invention also relates to recombinant vectors which includethe isolated nucleic acid molecules of the present invention, host cellscontaining the recombinant vectors, and the production of T1R-likeligand II polypeptides or fragments thereof by recombinant techniques.

The invention further provides an isolated T1R-like ligand IIpolypeptide having an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of the T1R-like ligand IIpolypeptide having the complete 229 amino acid sequence, including theleader sequence shown in SEQ ID NO:2; (b) the amino acid sequence of theT1R-like ligand II polypeptide having the complete 229 amino acidsequence, including the leader sequence shown in SEQ ID NO:2 but minusthe N-terminal methionine residue; (c) the amino acid sequence of themature T1R-like ligand II polypeptide (without the leader) having theamino acid sequence at positions 1 to 203 in SEQ ID NO:2; (d) the aminoacid sequence of the T1R-like ligand II polypeptide having the completeamino acid sequence, including the leader, encoded by the cDNA clonecontained in ATCC Deposit No. 97655; and (e) the amino acid sequence ofthe mature T1R-like ligand II polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97655. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 90% identical, and more preferably 95%,96%, 97%, 98% or 99% identical to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a T1R-like ligand II polypeptide having anamino acid sequence described in (a), (b), (c), (d), or (e), above.Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a T1R-like ligand II polypeptide of theinvention include portions of such polypeptides with at least six orseven, preferably at least nine, and more preferably at least about 30amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention. In another embodiment, the invention providesan isolated antibody that binds specifically to a T1R-like ligand IIpolypeptide having an amino acid sequence described in (a), (b), (c),(d), or (e) above.

The invention also relates to fragments of the above-describedpolypeptides. Preferred polypeptide fragments according to the presentinvention include a polypeptide comprising: the mature polypeptide(amino acid residues from about 1 to about 203 in SEQ ID NO:2), theextracellular domain (amino acid residues from about 1 to about 168 inSEQ ID NO:2), the transmembrane domain (amino acid residues from about169 to about 191 in SEQ ID NO:2), the intracellular domain (amino acidresidues from about 192 to about 203 in SEQ ID NO:2), or theextracellular and intracellular domain with all or part ofthetransmembrane domain deleted.

In addition, the invention provides for fusion polypeptides of T1R-likeligand II which maybe generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling and/or codon-shuffling.

The invention further provides for proteins containing polypeptidesequences encoded by the polynucleotides of the invention. The proteinsmay be in the form of monomers or multimers. The preparation of theseproteins and compositions (preferably pharmaceutical compositions)containing these proteins are also provided.

In another embodiment, the invention provides transgenic animals whichexpress the polypeptides and proteins of the invention.

In yet another embodiment, chromosome assays are provided which allowfor chromosome identification. Nucleic acids of the invention can beused to specifically target and hybridize to a particular location on anindividual human chromosome. Once a sequence has been mapped to aprecise chromosome location, the physical position of the sequence onthe chromosome can be correlated with genetic map data.

In another embodiment, the invention provides for antisense and ribozymeantagonists of T1R-like ligand II.

It is believed that biological activities ofthe T1R-like ligand II ofthepresent invention may be similar to the biological activities of the T1Rligand and IL-1. Significantly, higher or lower levels of T1R-likeligand II maybe detected in tissues or bodily fluids (e.g., serum,plasma, urine, synovial fluid or spinal fluid) taken from an individualhaving a T1R ligand- or IL-1-related disorder, relative to a “normal”T1R-like ligand II gene expression level, i.e., the expression level intissue or bodily fluids from an individual not having the T1R ligand- orIL-1-related disorder. Thus, detecting expression of T1R-like ligand IIgene expression according to the present invention is a diagnosticmarker. Accordingly, the invention provides for diagnostic kits used todetect levels of T1R-like ligand II expression.

The invention also provides methods for producing and isolatingantibodies that bind specifically to an T1R-like ligand II polypeptidehaving an amino acid sequence as described herein. Such antibodies areuseful diagnostically or therapeutically as described herein.

The invention is further related to a method for treating an individualin need of an increased or decreased level of T1R-like ligand IIactivity in the body, comprising administering to such an individual acomposition comprising a T1R-like ligand II polypeptide or an inhibitorthereof.

As such, pharmaceutical compositions of T1R-like ligand II are provided.Formulations of T1R-like ligand II are also provided as are methods foradministering therapeutic doses of T1R-like ligand II polynucleotides,polypeptides, antibodies, agonists, antagonists and/or fragments andvariants thereof.

Finally, the invention provides for methods of using the polynucleotidesencoding T1R-like ligand II polypeptides, antibodies, agonists,antagonists, and/or fragments and variants thereof, in gene therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–B shows the nucleotide (SEQ ID NO:1) and deduced amino acid(SEQ ID NO:2) sequences of the T1R-like ligand II protein determined bysequencing the cDNA clone contained in ATCC Deposit No. 97655. Theprotein has a leader sequence of about 26 amino acid residues (firstunderlined sequence), an extracellular mature domain of about 168 aminoacid residues (sequence between the first and second underlinedsequences), a transmembrane domain of about 23 amino acid residues(second underlined sequence), and an intracellular domain of about 12amino acid residues (the remaining sequence).

FIG. 2 shows the regions of similarity between the amino acid sequencesof the T1R-like ligand II and the protein sequence of GenBank accessionNo. U41804 (SEQ ID NO:3), showing an overall 56% identity.

FIG. 3 provides an analysis of the T1R-like ligand II amino acidsequence. Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown.

FIG. 4 shows the effect of T1R-like ligand II containing supernatant onCD34+Bone Marrow Proliferation Assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated nucleic acid moleculecomprising a polynucleotide encoding a T1R-like ligand II protein havingan amino acid sequence shown in FIGS. 1A–B (SEQ ID NO:2), which wasdetermined by sequencing a cloned cDNA. The T1R-like ligand II proteinof the present invention shares sequence homology with the T1R ligand(SEQ ID NO:3).

The nucleotide sequence in FIGS. 1A–B (SEQ ID NO:1) was obtained bysequencing the HE9BK24 clone, which was deposited on Jul. 12, 1996 atthe American Type Culture Collection, Patent Depository, 10801University Boulevard, Manassas, Va. 20110–2209, and given accessionnumber 97655. The deposited clone is contained in the pBluescript SK(−)plasmid (Stratagene, LaJolla, Calif.).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of peptide, polypeptides or proteins encoded by DNAmolecules determined herein were expected by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein can contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule.

The actual sequence can be more precisely determined by other approachesincluding manual DNA sequencing methods well known in the art. As isalso known in the art, a single insertion or deletion in a determinednucleotide sequence compared to the actual sequence will cause a frameshift in translation of the nucleotide sequence such that the expectedamino acid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U) where each thymidine deoxynucleotide (T) in the specifieddeoxynucleotide sequence is replaced by the ribonucleotide uridine (U).For instance, reference to an RNA molecule having the sequence in SEQ IDNO:1 set forth using deoxyribonucleotide abbreviations is intended toindicate an RNA molecule having a sequence in which each deoxynucleotideA, G or C in SEQ ID NO:1 has been replaced by the correspondingribonucleotide A, G or C, and each deoxynucleotide T has been replacedby a ribonucleotide U.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes ofthe present invention.Further examples of isolated DNA molecules include recombinant DNAmolecules maintained in heterologous host cells or purified (partiallyor substantially) DNA molecules in solution. Isolated RNA moleculesinclude in vivo or in vitro RNA transcripts of the DNA molecules of thepresent invention. Isolated nucleic acid molecules according to thepresent invention further include such molecules produced synthetically.

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A–B (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding an T1R-like ligand II polypeptide can be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIGS. 1A–B (SEQ IDNO:1) was discovered in a cDNA library derived from nine week old humanembryo tissue. Further, the gene was also found in cDNA librariesderived from the following types of human cells: prostate, anergicT-cell, TF274 stromal, WI 38, Soares breast, and Soares placenta.

The T1R-like ligand II cDNA contains an open reading frame encoding aprotein of about 229 amino acid residues whose initiation codon is atpositions 55–57 of the nucleotide sequence shown in SEQ ID NO. 1; apredicted leader sequence of about 26 amino acid residues and a deducedmolecular weight of about 26 kDa. The amino acid sequence of the matureT1R-like ligand II protein is shown in SEQ ID NO:2 from amino acidresidue 1 to residue 203. The mature T1R-like ligand II protein hasthree main structural domains. These include the extracellular domain,from amino acid residue about 1 to about 168 in SEQ ID NO:2; thetransmembrane domain, from amino acid residue about 169 to about 191 inSEQ ID NO:2; and the intracellular domain, from amino acid residue about192 to about 203 in SEQ ID NO:2. The T1R-like ligand II protein of thepresent invention in SEQ ID NO:2 is about 56% identical and about 75%similar to the T1R ligand, which can be accessed on GenBank as AccessionNo. U41804.

As indicated, the present invention also provides the mature form(s) ofthe T1R-like ligand II protein of the present invention. According tothe signal hypothesis, proteins secreted by mammalian cells have asignal or secretory leader sequence which is cleaved from the matureprotein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species on the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature T1R-like ligand II polypeptideshaving the amino acid sequence encoded by the cDNA clone contained inthe host identified as ATCC Deposit No. 97655 and as shown in SEQ IDNO:2. By the mature T1R-like ligand II protein having the amino acidsequence encoded by the cDNA clone contained in the host identified asATCC Deposit 97655 is meant the mature form(s) of the T1R-like ligand IIprotein produced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humanDNA sequence of the clone contained in the vector in the deposited host.As indicated below, the mature T1R-like ligand II having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97655may or may not differ from the predicted “mature” T1R-like ligand IIprotein shown in SEQ ID NO:2 (amino acids from about 1 to about 203)depending on the accuracy of the predicted cleavage site based oncomputer analysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available because itis known that much of the cleavage specificity for a secretory proteinresides in certain amino acid residues within the signal sequence andthe N-terminus of the mature protein, particularly residues immediatelysurrounding the cleavage site. For instance, the method of McGeoch(Virus Res. 3:271–286 (1985)) uses the information from a shortN-terminal charged region and a subsequent uncharged region of thecomplete (uncleaved) protein. The method of von Heinje (Nucleic AcidsRes. 14:4683–4690 (1986)) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2 where +1indicates the amino acid terminus ofthe mature protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75–80%. von Heinje, supra.However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, as well as the variability ofcleavage sites for leaders in different known proteins, the actualT1R-like ligand II encoded by the deposited cDNA comprises about 229amino acids, but can be anywhere in the range of 215–245 amino acids;and the deduced leader sequence of this protein is about 26 amino acids,but can be anywhere in the range of about 15 to about 30 amino acids.Further, for example, the exact locations ofthe T1R-like ligand IIprotein extracellular, intracellular and transmembrane domains in SEQ IDNO:2 may vary slightly (e.g., the exact amino acid positions may differby about 1 to about 5 residues compared to that shown in SEQ ID NO:2)depending on the criteria used to define the domain.

As indicated, nucleic acid molecules of the present invention can be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA can be double-stranded or single-stranded.Single-stranded DNA or RNA can be the coding strand, also known as thesense strand, or it can be the non-coding strand, also referred to asthe anti-sense strand.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 55–57 of the nucleotide sequence shown in FIGS. 1A–B(SEQ ID NO:1) and further include DNA molecules which comprise asequence substantially different that all or part of the ORF whoseinitiation codon is at position 55–57 of the nucleotide sequence inFIGS. 1A–B (SEQ ID NO:1) but which, due to the degeneracy of the geneticcode, still encode the T1R-like ligand II protein or a fragment thereof.Of course, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate the degenerate variantsdescribed above.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the T1R-like ligand II protein having an amino acidsequence encoded by the cDNA clone contained in the plasmid deposited asATCC Deposit No. 97655 on Jul. 12, 1996. Preferably, this nucleic acidmolecule will encode the mature polypeptide encoded by theabove-described deposited cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIGS. 1A–B (SEQ ID NO:1) or thenucleotide sequence of the T1R-like ligand II cDNA contained in theabove-described deposited clone, or having a sequence complementary toone of the above sequences. Such isolated molecules, particularly DNAmolecules, are useful as probes for gene mapping by in situhybridization with chromosomes and for detecting expression of theT1R-like ligand II gene in human tissue, for instance, by Northern blotanalysis. As described in detail herein, detecting altered T1R-likeligand II gene expression in certain tissues may be indicative ofcertain disorders.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having, for example, the nucleotide sequence ofthe deposited cDNA ATCC No. 97655, a nucleotide sequence encoding thepolypeptide sequence encoded by the deposited cDNA, a nucleotidesequence encoding the polypeptide sequence depicted in FIGS. 1A–B (SEQID NO:2), the nucleotide sequence shown in FIGS. 1A–B (SEQ ID NO:1), orthe complementary strand thereto, is intended fragments at least 15 nt,and more preferably at least about 20 nt, still more preferably at least30 nt, and even more preferably, at least about 40, 50, 100, 150, 200,250, 300, 325, 350, 375,400, 450, 500, 550, 600 or 650 nt in length.These fragments have numerous uses which include, but are not limitedto, diagnostic probes and primers as discussed herein. Of course, largerfragments, such as those of 700–1244 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequences of the deposited cDNA ATCCNo. 97655 or as shown in FIGS. 1A–B (SEQ ID NO:1). By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from, for example, the nucleotide sequenceof the deposited cDNA, or the nucleotide sequence as shown in FIGS. 1A–B(SEQ ID NO:1).

Since the gene has been deposited and the nucleotide sequence shown inFIGS. 1A–B (SEQ ID NO:1) is provided, generating such DNA fragmentswould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication could easily be used togenerate fragments of various sizes. Alternatively, such fragments couldbe generated synthetically.

Representative examples of T1R-like ligand II polynucleotide fragmentsof the invention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to 50, 51to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 600 to 650, 651to 700, 701 to 750, 751 to 800, 800 to 850, 851 to 900, 901 to 950, 951to 1000, 1001 to 1050, 1051 to 1100, 1101 to 1150, and/or 1151 to 1210of SEQ ID NO:1, or the complementary strand thereto, or the cDNAcontained in the deposited plasmid. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising the T1R-likeligand II extracellular domain (amino acid residues from about 1 toabout 168 in SEQ ID NO:2); a polypeptide comprising the T1R-like ligandII transmembrane domain (amino acid residues from about 169 to about 191in SEQ ID NO:2); a polypeptide comprising the T1R-like ligand IIintracellular domain (amino acid residues from about 192 to about 203 inSEQ ID NO:2); and a polypeptide comprising the T1R-like ligand IIextracellular and intracellular domains having all or part of thetransmembrane domain deleted. Further preferred nucleic acid fragmentsof the present invention include nucleic acid molecules encodingepitope-bearing portions of the T1R-like ligand II protein. Inparticular, isolated nucleic acid molecules are provided encodingpolypeptides comprising the following amino acid residues in SEQ IDNO:2, which the present inventors have determined are hydrophilicregions of the T1R-like ligand II protein: a polypeptide comprisingamino acid residues from about 17 to about 26 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 56 to about 72 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 103to about 120 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 136 to about 149 in SEQ ID NO:2; and a polypeptidecomprising amino acid residues from about 155 to about 171 in SEQ IDNO:2. Methods for determining other such epitope-bearing portions of theT1R-like ligand II protein are described in detail herein.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a T1R-like ligand II functional activity.By a polypeptide demonstrating a T1R-like ligand II “functionalactivity” is meant, a polypeptide capable of displaying one or moreknown functional activities associated with a full-length (complete) orsoluble T1R-like ligand II protein. Such functional activities include,but are not limited to, biological activity (e.g., ability to regulate(e.g., stimulate) hematopoiesis in vitro or in vivo), antigenicity[ability to bind (or compete with a T1R-like ligand II polypeptide forbinding) to an anti-T1R-like ligand II antibody], immunogenicity(ability to generate antibody which binds to a T1R-like ligand IIpolypeptide), ability to form multimers with T1R-like ligand IIpolypeptides of the invention, and ability to bind to a receptor orligand for a T1R-like ligand II polypeptide.

The functional activity of T1R-like ligand II polypeptides, andfragments, variants derivatives, and analogs thereof, can be assayed byvarious methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length T1R-like ligand II polypeptide forbinding to anti-T1R-like ligand II antibody, various immunoassays knownin the art can be used, including but not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where a T1R-like ligand II polypeptide ligand isidentified, or the ability of a polypeptide fragment, variant orderivative ofthe invention to multimerize is being evaluated, bindingcan be assayed, e.g., by means well-known in the art, such as, forexample, reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., 1995, Microbiol. Rev. 59:94–123. In another embodiment,physiological correlates of T1R-like ligand II binding to its substrates(signal transduction) can be assayed.

In addition, assays described herein and otherwise known in the art mayroutinely be applied to measure the ability of T1R-like ligand IIpolypeptides and fragments, variants derivatives and analogs thereof toelicit T1R-like ligand II related biological activity [e.g., to regulate(e.g., to stimulate or inhibit) hematopoiesis in vitro or in vivo]. Forexample, techniques known in the art (such as for example assaying forthymidine incorporation), may be applied or routinely modified to assayfor the ability of the compositions ofthe invention to inhibitproliferation of hematopoietic cells.

Other methods will be known to the skilled artisan and are within thescope of the invention.

In addition, the present inventors have identified nucleic acidmolecules having nucleotide sequences related to extensive portions ofSEQ ID NO:1 which have been determined from the following related cDNAclone: HPVAA83R (SEQ ID NO:11).

The following public ESTs are related to extensive portions of SEQ IDNO:1: GenBank accession No. AA013099 (SEQ ID NO:12), GenBank accessionNo. AA251084 (SEQ ID NO:13), GenBank accession No. R58562 (SEQ IDNO:14), GenBank accession No. N28878 (SEQ ID NO:15), GenBank accessionNo. AA019348 (SEQ ID NO:16), GenBank accession No. N49615 (SEQ IDNO:17), GenBank accession No. AA112675 (SEQ ID NO:18), GenBank accessionNo. AA082161 (SEQ ID NO:19), GenBank accession No. H03613 (SEQ IDNO:20), GenBank accession No. R54717 (SEQ ID NO:2 1), GenBank accessionNo. H27167 (SEQ ID NO:22), GenBank accession No. AA188741 (SEQ IDNO:23), GenBank accession No. AA094735 (SEQ ID NO:24) and GenBankaccession No. AA285143 (SEQ ID NO:25).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule ofthe invention described above, for instance, the cDNAclone contained in ATCC Deposit 97655. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. By a polynucleotide whichhybridizes to a “portion” of a polynucleotide is intended apolynucleotide (either DNA or RNA) hybridizing to at least about 15nucleotides (nt), and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably at least about30–70 nt of the reference polynucleotide. These are useful as diagnosticprobes and primers as discussed above and in more detail herein.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650nt in length, or even to the entire length of the referencepolynucleotide, also are useful as probes according to the presentinvention, as are polynucleotides corresponding to most, if not all, ofthe nucleotide sequence ofthe deposited cDNA or the nucleotide sequenceas shown in FIGS. 1A–B (SEQ ID NO:1). By a portion of a polynucleotideof “at least 20 nt in length, ” for example, is intended 20 or morecontiguous nucleotides from the nucleotide sequence of the referencepolynucleotide, (e.g., the deposited cDNA or the nucleotide sequence asshown in FIGS. 1A–B (SEQ ID NO:1)). As indicated, such portions areuseful diagnostically either as a probe according to conventional DNAhybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Sambrook, J. et al., eds., Molecular Cloning, A LaboratoryManual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

Since an T1R-like ligand II cDNA clone has been deposited and itsdetermined nucleotide sequence is provided in FIGS. 1A–B (SEQ ID NO:1),generating polynucleotides which hybridize to a portion of the T1R-likeligand II cDNA molecule would be routine to the skilled artisan. Forexample, restriction endonuclease cleavage or shearing by sonication ofthe T1R-like ligand II cDNA clone could easily be used to generate DNAportions of various sizes which are polynucleotides that hybridize to aportion of the T1R-like ligand II cDNA molecule. Alternatively, thehybridizing polynucleotides of the present invention could be generatedsynthetically according to known techniques.

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the T1R-like ligand II cDNAshown in FIGS. 1A–B (SEQ ID NO:1)), or to a complementary stretch of T(or U) resides, would not be included in a polynucleotide of theinvention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule contain a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode the T1R-like ligand II can include, but are not limited to, thoseencoding the amino acid sequence of the mature polypeptide, by itself,the coding sequence for the mature polypeptide and additional sequences,such as those encoding the about 26 amino acid leader sequence, such asa pre-, or pro- or prepro-protein sequence; the coding sequence of themature polypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, e.g., ribosome binding and stability of mRNA; an additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, the sequence encodingthe polypeptide can be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are publicly and/or commercially available. As describedin Gentz et al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. The “HA” tag is another peptide useful for purificationwhich corresponds to an epitope derived from the influenza hemagglutinin(HA) protein, which has been described by Wilson et al., Cell 37:767(1984). Other such fusion proteins include the T1R-like ligand IIprotein or a fragment thereof fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the T1R-like ligand II protein. Variants can occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. Non-naturally occurring variantsmay be produced using art-known mutagenesis techniques, which include,but are not limited to oligonucleotide mediated mutagenesis, alaninescanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carteret al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. AcidsRes. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al., Gene34:315 (1985)), restriction selection mutagenesis (see e.g., Wells etal., Philos. Trans. R. Soc. London SerA 317:415 (1986)).

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions caninvolve one or more nucleotides. The variants can be altered in codingor non-coding regions or both. Alterations in the coding regions canproduce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the T1R-like ligand II or portions thereof.Also especially preferred in this regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 203 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97655; (e) a nucleotidesequence encoding the mature T1R-like ligand II polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97655; or (f) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), or (e).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a T1R-likeligand II polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five mutations per each 100nucleotides of the reference nucleotide sequence encoding the T1R-likeligand II polypeptide. In other words, to obtain a polynucleotide havinga nucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence can bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mismatches of thereference sequence can occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entireT1R-like ligand II encoding nucleotide sequence shown in FIGS. 1A–B (SEQID NO:1) or any T1R-like ligand II polynucleotide fragment (e.g., apolynucleotide encoding the amino acid sequence of any of the T1R-likeligand II—and/or C-terminal deletions described herein), variant,derivative or analog, as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theencoding nucleotide sequence shown in FIGS. 1A–B (SEQ ID NO:1), or tothe nucleotide sequence of the deposited cDNA plasmid, can be determinedconventionally using known computer programs such as the BESTFIT™program (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science Drive,Madison, Wis. 53711). BESTFIT™ uses the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2:482–489 (1981), tofind the best segment of homology between two sequences. When usingBESTFIT™ or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237–245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap SizePenalty 0.05, Window Size-500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.

A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%.

In another example, a 90 base subject sequence is compared with a 100base query sequence. This time the deletions are internal deletions sothat there are no bases on the 5′ or 3′ of the subject sequence whichare not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only bases 5′ and 3′ of the subject sequence which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesdisclosed herein, (e.g., encoding a polypeptide having the amino acidsequence of an N and/or C terminal deletion disclosed herein, such as,for example, a nucleic acid molecule encoding amino acids −26 to 203 ofSEQ ID NO:2), irrespective of whether they encode a polypeptide havingT1R-like ligand II functional activity. This is because even where aparticular nucleic acid molecule does not encode a polypeptide havingT1R-like ligand II functional activity, one of skill in the art wouldstill know how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer.

Uses of the nucleic acid molecules of the present invention that do notencode a polypeptide having T1R-like ligand II functional activityinclude, inter alia, (1) isolating a T1R-like ligand II gene or allelicor splice variants thereof in a cDNA library; (2) in situ hybridization(e.g., “FISH”) to metaphase chromosomal spreads to provide precisechromosomal location of the T1R-like ligand II gene, as described inVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988); and (3) Northern Blot analysis for detectingT1R-like ligand II mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesdisclosed herein, which do, in fact, encode a polypeptide havingT1R-like ligand II functional activity. By “a polypeptide havingT1R-like ligand II functional activity” is intended polypeptidesexhibiting activity similar, but not necessarily identical, to afunctional activity of the T1R-like ligand II polypeptides of thepresent invention (e.g., complete (full-length) T1R-like ligand II,mature T1R-like ligand II and soluble T1R-like Ligand II (e.g., havingsequences contained in the extracellular domain of T1R-like ligand II)as measured, for example, in a particular immunoassay or biologicalassay. For example, a T1R-like ligand II functional activity canroutinely be measured by determining the ability of a T1R-like ligand IIpolypeptide to bind a T1R-like ligand II ligand. T1R-like ligand IIfunctional activity may also be measured by determining the ability of apolypeptide, such as cognate ligand which is free or expressed on a cellsurface, to induce hematopoiesis in cells expressing the polypeptide.

T1R-like ligand II activity can be further assayed using known receptorbinding assays (Mitcham, J. L. et al., J. Biol. Chem. 271:5777–5783(1996); and Gayle, M. A. et al., J. Biol. Chem. 271:5784–5789 (1996)).These assays include an NF-κB gel shift assay, an in vitro Thr-669kinase assay, and an IL-8 promoter activation assay.

To perform these assays, it is first necessary to transfect mammaliancells with an expression vector containing the cDNA for a suitablereceptor. For example, an expression vector containing the cDNA for theT1/ST2 receptor can be used. This cDNA can be obtained as described(Klemenz, R. et al., Proc, Natl. Acad. Sci. U.S.A. 86:5708–5712 (1989);Tominaga, S., FEBS Lett. 258:301–304; Bergers, G. et al. EMBO J.13:1176–1188)). Alternatively, T1/ST2 cDNA can be amplified using thepolymerase chain reaction. A commercially available cDNA library,prepared from mRNA from a suitable tissue or cell type (such as NIH-3T3cells (Klemenz, R. et al., Proc, Natl. Acad. Sci. U.S.A. 86:5708–5712(1989)), can be used as template. Using any of several transfectionmethods well known to those of ordinary skill in the art, a suitablecell line (e.g., COS 7 cells) can be transfected with the T1/ST2expression plasmid. Expression ofthe receptor can be verified byradioimmunoassay (see Mitcham, J. L. et al., J. Biol. Chem.271:5777–5783 (1996)). One to three days post-transfection, confluenttransfected COS7 cells are stimulated with 1–10 ng of T1R-like ligand IIprotein for 15 minutes to 20 hours. Duration of stimulation by T1R-likeligand II protein will vary, depending on which assay is used, and canbe determined using only routine experimentation.

To perform the NF-κB assay, nuclear extracts from transfected cells areprepared immediately after stimulation (Ostrowski, J. et al., J. Biol.Chem. 266: 12722–12733 (1991)). A double-stranded syntheticoligonucleotide probe containing the NF-κB enhancer element from theimmunoglobulin K light chain is 5′-end labeled by phosphorylation with[γ-³²P]ATP (5′ TGACAGAGGGACTTTCCGAGAGGA 3′ (SEQ ID NO:10)). Nuclearextracts (10 μg) are incubated with radiolabeled probe for 20 minutes atroom temperature, and protein-DNA complexes are resolved byelectrophoresis in a 0.5× TBE, 10% polyacrylamide gel.

To perform the in vitro Thr-669 kinase assay, cytoplasmic extracts oftransfected cells are prepared immediately after stimulation (Bird, T.A. et al., Cytokine 4:429–440 (1992)). 10 μl of cell extract is added to20 μl of reaction mixture containing 20 mM HEPES buffer (pH 7.4), 15 mMMgCl₂, 15 μM ATP, 75 μCi/ml [γ-³²P]ATP, and 750 μM substrate peptide(residues 663–673 of EGFR). Blanks are incubated with distilled H₂O in,place of the peptide. After incubation at 30° C. for 20 minutes, thereactions are terminated by addition of formic acid. Reactions arecleared by centrifugation, and 30 μl of supernatant are spotted onphosphocellulose paper discs. After washing (three times with 75 mMorthophosphoric acid) and drying, peptide-incorporated counts aredetermined by monitoring Cerenkov counts. Results are expressed as theratio of Thr-669 kinase activity detected in nonstimulated cellscompared to activity detected in stimulated cells.

To perform the IL-8 promoter activation assay, COS7 cells (1×10⁵ cellsper well in a multi-well tissue culture plate) are cotransfected withthe T1/ST2 receptor expression vector and the pIL8p reporter plasmid(Mitcham, J. L. et al., J. Biol. Chem. 271:5777–5783 (1996)). One daypost-transfection, the medium is changed and cells are either stimulatedwith 1 ng/ml IL-1α or are left stimulated. 12–16 hours post-stimulation,cells are washed twice with binding medium containing 5% (w/v) non-fatdry milk (5% MBM) and blocked with 2 ml of 5% MBM at room temperaturefor 30 minutes. Cells are then incubated at room temperature for 60–90minutes with 1.5 ml/well of 5% MBM containing 1 μg/ml of an anti-IL-2Rαantibody (R&D Systems, Minneapolis, Minn.) with gentle rocking. Cellsare washed once with 5% MBM and incubated with 1 ml/well of 5% MBMcontaining 1:100 dilution of ¹²⁵I-goat anti-mouse IgG (Sigma, St. Louis,Mo.) for 60 minutes at room temperature. Wells are washed four timeswith 5% MBM and twice with phosphate-buffered saline. Wells are strippedby the addition of 1 ml of 0.5 M NaOH, and total counts are determined.Results are expressed as total cpm averaged over two duplicate or threetriplicate wells.

Thus, “a polypeptide having T1R-like ligand II protein activity”includes polypeptides that exhibit T1R-like ligand II protein activityin at least one of the above-described assays.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the depositedcDNA, the nucleic acid sequence shown in FIGS. 1A–B (SEQ ID NO:1), orfragments thereof, will encode polypeptides “having T1R-like ligand IIfunctional activity.” In fact, since degenerate variants of any of thesenucleotide sequences all encode the same polypeptide, in many instances,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingT1R-like ligand II functional activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid),as further described herein.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306–1310 (1990), wherein the authors indicate that there are twomain approaches for studying the tolerance of an amino acid sequence tochange. The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J.U., et al., supra, and the references cited therein.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of T1R-likeligand II polypeptides or fragments thereof by recombinant techniques.

Recombinant constructs may be introduced into host cells using wellknown techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include bacterial cells,such as E. coli, Streptomyces and Salmonella typhimurium cells; fungalcells, such as yeast cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanomacells; and plant cells. Appropriate culture media and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG andpSVL available from Pharmacia, and pA2 available from Qiagen. Othersuitable vectors will be readily apparent to the skilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and met allothionein promoters, such as the mousemet allothionein-I promoter.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods in MolecularBiology (1986).

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., T1R-like ligand II coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with T1R-like ligand II polynucleotides ofthe invention, and which activates, alters, and/or amplifies endogenousT1R-like ligand II polynucleotides. For example, techniques known in theart may be used to operably associate heterologous control regions(e.g., promoter and/or enhancer) and endogenous T1R-like ligand IIpolynucleotide sequences via homologous recombination (see, e.g., U.S.Pat. Number 5, 641, 670; International Publication Number WO 96/29411;International application publication number WO 94/12650; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932–8935 (1989); and Zijlstra et al.,Nature 342:435–438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

In one embodiment, polynucleotides encoding T1R-like ligand IIpolypeptides of the invention may be fused to the pelB pectate lyasesignal sequence to increase the efficiency to expression andpurification of such polypeptides in Gram-negative bacteria. See, U.S.Pat. Nos. 5,576,195 and 5,846,818, the contents of which are hereinincorporated by reference in their entireties.

Thus, the polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins.

For example, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5—has been fused with Fc portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. See, D. Bennett etal., Journal of Molecular Recognition, Vol. 8 52–58 (1995) and K.Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16,pp 9459–9471 (1995).

The T1R-like ligand II can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification.

In addition, proteins of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., andHunkapiller, M., et al., Nature 310:105–111 (1984)). For example, apeptide corresponding to a fragment of the T1R-like ligand IIpolypeptides ofthe invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the T1R-like ligand II polypeptide sequence. Non-classicalamino acids include, but are not limited to, to the D-isomers of thecommon amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The T1R-like ligand II proteins of the invention may be modified byeither natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given T1R-like ligandII polypeptide. T1R-like ligand II polypeptides may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic T1R-like ligandII polypeptides may result from posttranslation natural processes or maybe made by synthetic methods.

Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1–12 (1983); Seifter et al., Meth Enzymol 182:626–646(1990); Rattan et al., Ann NY Acad Sci 663:48–62 (1992).)

The invention additionally, encompasses T1R-like ligand II polypeptideswhich are differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, iodination,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, etc.Any of numerous chemical modifications may be carried out by knowntechniques, including but not limited to, specific chemical cleavage bycyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄,acetylation, formylation, oxidation, reduction, metabolic synthesis inthe presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends, attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofT1R-like ligand II which may provide additional advantages such asincreased solubility, stability and circulating time of the polypeptide,or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028–1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

Thus, polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

Polypeptides and Peptides of T1R-like Ligand II

The invention further provides an isolated T1R-like ligand IIpolypeptide having the amino acid sequence encoded by the depositedcDNA, or the amino acid sequence in FIGS. 1A–B (SEQ ID NO:2), or apeptide or polypeptide comprising a portion of the above polypeptides.The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably as thecontext requires to indicate a chain of at least two amino acids coupledby peptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than ten amino acid residues. All oligopeptide andpolypeptide formulas or sequences herein are written from left to rightand in the direction from amino terminus to carboxy terminus.

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for purposes of the invention as are native orrecombinant polypeptides and proteins which have been substantiallypurified by any suitable technique such as, for example, the one-stepmethod described in Smith and Johnson, Gene 67:31–40 (1988).

It will be recognized in the art that some amino acid sequence oftheT1R-like ligand II can be varied without significant effect on thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of the T1R-like ligandII which show substantial T1R-like ligand II activity or which includeregions of T1R-like ligand II such as the protein portions discussedherein. Such mutants include deletions, insertions, inversions, repeats,and type substitutions (for example, substituting one hydrophilicresidue for another, but not strongly hydrophilic for stronglyhydrophobic as a rule). Small changes or such “neutral” amino acidsubstitutions will generally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr.

As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions, ” Science247:1306–1310 (1990).

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more oftheamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics ofthe T1R-like ligand II protein.

The prevention of aggregation is highly desirable. Aggregation ofproteins not only results in a loss of activity but can also beproblematic when preparing pharmaceutical formulations, because they canbe immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331–340 (1967);Robbins et al., Diabetes 36:838–845 (1987); Cleland et al. Crit. Rev.Therapeutic Drug Carrier Systems 10:307–377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266–268(1993) describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, theT1R-like ligand II of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given PAPAIpolypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3.

Amino acids in the T1R-like ligand II protein of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081–1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as receptor binding or in vitro, or in vivoproliferative activity. Sites that are critical for ligand-receptorbinding can also be determined by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith et al., J. Mol. Biol. 224:899–904 (1992) and de Vos et al.Science 255:306–312 (1992)).

The polypeptides ofthe present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell or a native source. Forexample, a recombinantly produced version of the T1R-like ligand IIpolypeptide can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67:31–40 (1988).

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein); a polypeptide comprising amino acids about −26 toabout 203 in SEQ ID NO:2; a polypeptide comprising amino acids about −25to about 203 in SEQ ID NO:2; a polypeptide comprising amino acids about1 to about 203 in SEQ ID NO:2; as well as polypeptides at least 90%identical, and more preferably at least 95%, 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA, to thepolypeptide of SEQ ID NO: 2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a T1R-like ligandII polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of a T1R-like ligandII. In other words, to obtain a polypeptide having an amino acidsequence at least 95% identical to a reference amino acid sequence, upto 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A–B (SEQ ID NO:2), the amino acid sequenceencoded by the deposited cDNA clone, or fragments thereof, can bedetermined conventionally using known computer programs such theBESTFIT™ program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using BESTFIT™ or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237–245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residuesofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal rsidues of the subject sequence. For example, a 90 amino acidresidue subject sequence is aligned with a 100 residue query sequence todetermine percent identity. The deletion occurs at the N-terminus of thesubject sequence and therefore, the FASTDB alignment does not show amatching/alignment of the first 10 residues at the N-terminus. The 10unpaired residues represent 10% of the sequence (number of residues atthe N- and C-termini not matched/total number of residues in the querysequence) so 10% is subtracted from the percent identity scorecalculated by the FASTDB program. If the remaining 90 residues wereperfectly matched the final percent identity would be 90%. In anotherexample, a 90 residue subject sequence is compared with a 100 residuequery sequence. This time the deletions are internal deletions so thereare no residues at the N- or C-termini of the subject sequence which arenot matched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for. No other manualcorrections are made for the purposes of this embodiment.

In another embodiment of the present invention, there are providedfragments of the polypeptides described herein. Preferred fragmentsinclude: the extracellular domain (amino acid residues from about 1 toabout 168 in SEQ ID NO: 2); the transmembrane domain (amino acidresidues from about 169 to about 191 in SEQ ID NO: 2); the intracellulardomain (amino acid residues from about 192 to about 203 in SEQ ID NO:2); and the intracellular domain with all or part of the transmembranedomain deleted.

For many proteins, it is well known in the art that one or more aminoacids may be deleted from the N-terminus or C-terminus withoutsubstantial loss of biological function. However, even if deletion ofone or more amino acids from the N-terminus or C-terminus of a proteinresults in modification or loss of one or more biological functions ofthe protein, other T1R-like Receptor ligand functional activities (e.g.,biological activities (e.g., ability to regulate hematopoiesis), abilityto multimerize, ability to bind T1R-like ligand II polypeptide ligand)may still be retained. For example, the ability of shortened T1R-likeligand II mutants to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptides generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the N-terminus. Whether a particularpolypeptide lacking N-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that an T1R-like ligand II mutant with a large number ofdeleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the T1R-likeligand II amino acid sequence shown in FIGS. 1A–B (i.e., SEQ ID NO:2),up to the Lys residue at position number 198 and polynucleotidesencoding such polypeptides.

In particular, the present invention provides polypeptides comprisingthe amino acid sequence of residues n to 202 of SEQ ID NO:2, where n isan integer from 2 to 198 corresponding to the position of the amino acidresidue in SEQ ID NO:2. Preferably, N-terminal deletions of the T1R-likeligand II polypeptide of the invention shown as SEQ ID NO:2 includepolypeptides comprising, or alternatively consisting of, an amino acidsequence selected from amino acid residues: F-2 to T-203; T-3 to T-203;P-4 to T-203; S-5 to T-203; L-6 to T-203; D-7 to T-203; S-8 to T-203;D-9 to T-203; F-10 to T-203; T-11 to T-203; F-12 to T-203; T-13 toT-203; L-14 to T-203; P-15 to T-203; A-16 to T-203; G-17 to T-203; Q-18to T-203; K-19 to T-203; E-20 to T-203; C-21 to T-203; F-22 to T-203;Y-23 to T-203; Q-24 to T-203; P-25 to T-203; M-26 to T-203; P-27 toT-203; L-28 to T-203; K-29 to T-203; A-30 to T-203; S-31 to T-203; L-32to T-203; E-33 to T-203; I-34 to T-203; E-35 to T-203; Y-36 to T-203;Q-37 to T-203; V-38 to T-203; L-39 to T-203; D-40 to T-203; G-41 toT-203; A-42 to T-203; G-43 to T-203; L-44 to T-203; D-45 to T-203; I-46;to T-203; D-47 to T-203; F-48 to T-203; H-49 to T-203; L-50 to T-203;A-51 to T-203; S-52 to T-203; P-53 to T-203; E-54 to T-203; G-55 toT-203; K-56 to T-203; T-57 to T-203; L-58 to T-203; V-59 to T-203; F-60to T-203; E-61 to T-203; Q-62 to T-203; R-63 to T-203; K-64 to T-203;S-65 to T-203; D-66 to T-203; G-67 to T-203; V-68 to T-203; H-69 toT-203; T-70 to T-203; V-71 to T-203; E-72 to T-203; T-73 to T-203; E-74to T-203; V-75 to T-203; G-76 to T-203; D-77 to T-203; Y-78 to T-203;M-79 to T-203; F-80 to T-203; C-81 to T-203; F-82 to T-203; D-83 toT-203; N-84 to T-203; T-85 to T-203; F-86 to T-203; S-87 to T-203; T-88to T-203; I-89 to T-203; S-90 to T-203; E-91 to T-203; K-92 to T-203;V-93 to T-203; I-94 to T-203; F-95 to T-203; F-96 to T-203; E-97 toT-203; L-98 to T-203; I-99 to T-203; L-100 to T-203; D-101 to T-203;N-102 to T-203; M-103 to T-203; G-104 to T-203; E-105 to T-203; Q-106 toT-203; A-107 t T-203; Q-108 to T-203; E-109 to T-203; Q-110 to T-203;E-111 to T-203; D-112 to T-203; W-113 to T-203; K-114to T-203; K-115 toT-203; Y-116 to T-203; I-117 to T-203; T-118 to T-203; G-119 to T-203;T-120 to T-203; D-121 to T-203; I-122 to T-203; L-123 to T-203; D-124 toT-203; M-125 to T-203; K-126 to T-203; L-127 to T-203; E-128 to T-203;D-129 to T-203; I-130 to T-203; L-131 to T-203; E-132 to T-203; S-133 toT-203; I-134 to T-203; N-135 to T-203; S-136 to T-203; I-137 to T-203;K-138 to T-203; S-139 to T-203; R-140 to T-203; L-141 to T-203; S-142 toT-203; K-143 to T-203;S-144 to T-203; G-145 to T-203; H-146 to T-203;I-147 to T-203; Q-148 to T-203; T-149 to T-203; L-150 to T-203; L-151 toT-203; R-152 to T-203; A-153 to T-203; F-154 to T-203; E-155 to T-203;A-156 to T-203; R-157 to T-203; D-158 to T-203; R-159 to T-203; N-160 toT-203; I-161to T-203; Q-162 to T-203; E-163 to T-203; S-164 to T-203;N-165 to T-203; F-166 to T-203; D-167 to T-203; R-168 to T-203; V-169 toT-203; N-170 to T-203; F-171 to T-203; W-172 to T-203; S-173 to T-203;M-174 to T-203; V-175 to T-203; N-176 to T-203; L-177 to T-203; V-178 toT-203; V-179 to T-203; M-180 to T-203; V-181 to T-203; V-182 to T-203;V-183 to T-203; S-184 to T-203; A-185 to T-203; I-186 to T-203; Q-187 toT-203; V-188 to T-203; Y-189 to T-203; M-190 to T-203; L-191 to T-203;K-192 to T-203; S-193 to T-203; L-194 to T-203; F-195 to T-203; E-196 toT-203; D-197 to T-203; and K-198 to T-203; of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Additionally, N-terminal deletions of the T1R-like ligand II polypeptideof the invention shown as SEQ ID NO: 2 also include polypeptidescomprising, or alternatively consisting of, an amino acid sequenceselected from amino acid residues: F-2 to R-168; T-3 to R-168; P-4 toR-168; S-5 to R-168; L-6 to R-168; D-7 to R-168; S-8 to R-168; D-9 toR-168; F-10to R-168; T-11 to R-168; F-12 to R-168; T-13 to R-168; L-14toR-168; P-15 to R-168; A-16 to R-168; G-17 to R-168; Q-18 to R-168; K-19to R-168; E-20 to R-168; C-21 to R-168; F-22 to R-168; Y-23 to R-168;Q-24 to R-168; P-25 to R-168; M-26 to R-168; P-27 to R-168; L-28 toR-168; K-29 to R-168; A-30 to R-168; S-31 to R-168; L-32 to R-168; E-33to R-168; I-34 to R-168; E-35 to R-168; Y-36 to R-168; Q-37 to R-168;V-38 to R-168; L-39 to R-168; D-40 to R-168; G-41 to R-168; A-42 toR-168; G-43 to R-168; L-44 to R-168; D-45 to R-168; I-46 to R-168; D-47to R-168; F-48 to R-168; H-49 to R-168; L-50 to R-168; A-51 to R-168;S-52 to R-168; P-53 to R-168; E-54 to R-168; G-55 to R-168; K-56 toR-168; T-57 to R-168; L-58 to R-168; V-59 to R-168; F-60 to R-168; E-61to R-168; Q-62 to R-168; R-63 to R-168; K-64 to R-168; S-65 to R-168;D-66 to R-168; G-67 to R-168; V-68 to R-168; H-69 to R-168; T-70 toR-168; V-71 to R-168; E-72 to R-168; T-73 to R-168; E-74 to R-168; V-75to R-168; G-76 to R-168; D-77 to R-168; Y-78 to R-168; M-79 to R-168;F-80 to R-168; C-81 to R-168; F-82 to R-168; D-83 to R-168; N-84 toR-168; T-85 to R-168; F-86 to R-168; S-87 to R-168; T-88 to R-168; I-89to R-168; S-90to R-168; E-91 to R-168; K-92 toR-168; V-93 to R-168;I-94toR-168; F-95 to R-168; F-96 to R-168; E-97 to R-168; L-98 to R-168;I-99 to R-168; L-100to R-168; D-101 to R-168; N-102 to R-168; M-103 toR-168; G-104 to R-168; E-105 to R-168; Q-106 to R-168; A-107to R-168;Q-108 to R-168; E-109to R-168; Q-110to R-168; E-111 to R-168; D-112 toR-168; W-113 to R-168; K-114 to R-168; K-115 to R-168; Y-116 to R-168;I-117 to R-168, T-118 to R-168; G-119to R-168; T-120to R-168; D-121 toR-68; I-122 to R-168; L-123 to R-168; D-124 to R-168; M-125 to R-168;K-126 to R-168; L-127 to R-168; E-128 to R-168; D-129 to R-168; I-130 toR-168; L-131 to R-168; E-132 to R-168; S-133 to R-168; I-134 to R-168;N-135 to R-168; S-136 to R-168; I-137 to R-168; K-138 to R-168; S-139 toR-168; R-140 to R-168; L-141 to R-168; S-142 to R-168K-143 to R-168;S-144 to R-168; G-145 to R-168; H-146 to R-168; I-147 to R-168; Q-148 toR-168; T-149 to R-168; L-150 to R-168; L-151 to R-168; R-152 to R-168;A-153 to R-168; F-154 to R-168; E-155 to R-168; A-156 to R-168; R-157 toR-168; D-158 to R-168; R-159 to R-168; N-160 to R-168; I-161 to R-168;Q-162 to R-168; and E-163 to R-168; of SEQ ID NO:2. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to regulate hematopoiesis),ability to multimerize, ability to bind T1R-like ligand II polypeptideligand) may still be retained. For example the ability of the shortenedT1R-like ligand II mutant to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an T1R-like ligand II mutant with alarge number of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the T1R-like ligand II polypeptide shown in SEQ ID NO:2, upto the Leu residue at position number 6, and polynucleotides encodingsuch polypeptides.

In particular, the present invention provides polypeptides comprisingthe amino acid sequence of residues 1 to m of FIGS. 1A–B (i.e., SEQ IDNO:2), where m is an integer from 6 to 202 corresponding to the positionof the amino acid residue in SEQ ID NO:2.

Preferably, C-terminal deletions ofthe T1R-like ligand II polypeptide ofthe invention shown as SEQ ID NO: 2 include polypeptides comprising, oralternatively consisting of, an amino acid sequence selected from aminoacid residues: G-1 to R-202; G-1 to S-201; G-1 to K-200; G-1 to R-199;G-1 to K-198; G-1 to D-197; G-1 to E-196; G-1 to F-195; G-1 to L-194;G-1 to S-193; G-1 to K-192; G-1 to L-191; G-1 to M-190; G-1 to Y-189;G-1 to V-188; G-1 to Q-187; G-1 to I-186; G-1 to A-185; G-1 to S-184;G-1 to V-183; G-1 to V-182; G-1 to V-181; G-1 to M-180; G-1 to V-179;G-1 to V-178; G-1 to L-177; G-1 to N-176; G-1 to V-175; G-1 to M-174;G-1 to S-173; G-1 to W-172; G-1 to F-171; G-1 to N-170; G-1 to V-169;G-1 to R-168; G-1 to D-167; G-1 to F-166; G-1 to N-165; G-1 to S-164;G-1 to E-163; G-1 to Q-162; G-1 to I-161; G-1 to N-160; G-1 to R-159;G-1 to D-158; G-1 to R-157; G-1 to A-156; G-1 to E-155; G-1 to F-154;G-1 to A-153; G-1 to R-152; G-1 to L-151; G-1 to L-150; G-1 to T-149;G-1 to Q-148; G-1 to I-147; G-1 to H-146; G-1 to G-145; G-1 to S-144;G-1 to K-143; G-1 to S-142; G-1 to L-141; G-1 to R-140; G-1 to S-139;G-1 to K-138; G-1 to I-137; G-1 to S-136; G-1 to N-135; G-1 to I-134;G-1 to S-133; G-1 to E-132; G-1 to L-131; G-1 to I-130; G-1 to D-129;G-1 to E-128; G-1 to L-127; G-1 to K-126; G-1 to M-125; G-1 to D-124;G-1 to L-123; G-1 to I-122; G-1 to D-121; G-1 to T-120; G-1 to G-119;G-1 to T-118; G-1 to I-117; G-1 to Y-116; G-1 to K-115; G-1 to K-114;G-1 to W-113; G-1 to D-112; G-1 to E-111; G-1 to Q-110; G-1 to E-109;G-1 to Q-108; G-1 to A-107; G-1 to Q-106; G-1 to E-105; G-1 to G-104;G-1 to M-103; G-1 to N-102; G-1 to D-101; G-1 ; to L-100; G-1 to I-99;G-1 to L-98; G-1 to E-97; G-1 to F-96; G-1 to F-95; G-1 to I-94; G-1 toV-93; G-1 to K-92; G-1 to E-91; G-1 to S-90; G-1 to I-89; G-1 to T-88;G-1 to S-87; G-1 to F-86; G-1 to T-85; G-1 to N-84; G-1 to D-83; G-1 toF-82; G-1 to C-81; G-1 to F-80; G-1 to M-79; G-1 to Y-78; G-1 to D-77;G-1 to G-76; G-1 to V-75; G-1 to E-74; G-1 to T-73; G-1 to E-72; G-1 toV-71; G-1 to T-0; G-1 to H-69; G-1 to V-68; G-1 to G-67; G-1 to D-66;G-1 to S-65; G-1 to K-64; G-1 to R-63; G-1 to Q-62; G-1 to E-61; G-1 toF-60; G-1 to V-59; G-1 to L-58; G-1 to T-57; G-1 to K-56; G-1 to G-55;G-1 to E-54; G-1 to P-53; G-1 to S-52; G-1 to A-51; G-1 to L-50; G-1 toH-49; G-1 to F-48; G-1 to D-47; G-1 to I-46; G-1 to D-45; G-1 to L-44;G-1 to G-43; G-1 to A-42; G-1 to G-41; G-1 to D-40; G-1 to L-39; G-1 toV-38; G-1 to Q-37; G-1 to Y-36; G-1 to E-35; G-1 to I-34; G-1 to E-33;G-1 to L-32; G-1 to S-31; G-1 to A-30; G-1 to K-29; G1 to L-28; G-1 toP-27; G-1 to M-26; G-1 to P-25; G-1 to Q-24; G-1 to Y-23; G-1 to F-22;G-1 to C-21; G-1 to E-20; G-1 to K-19; G-1 to Q-18; G-1 to G-17; G-1 toA-16; G-1 to P-15; G-1 to L-14; G-1 to T-13; G-1 to F-12; G-1 to T-11;G-1 to F-10; G-1 to D-9; G-1 to S-8; G-1 to D-7; and G-1 to L-6; of SEQID NO:2. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of an T1R-likeligand II polypeptide, which may be described generally as havingresidues n-m of SEQ ID NO:2, where n and m are integers as describedabove.

The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to theT1R-like ligand II polypeptide sequence set forth herein as n to m. Inpreferred embodiments, the application is directed to proteinscontaining polypeptides at least 90%, 95%, 96%, 97%, 98% or 99%identical to polypeptides having the amino acid sequence of the specificT1R-like ligand II N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In certain preferred embodiments, T1R-like ligand II proteins of theinvention comprise fusion proteins as described above wherein theT1R-like ligand II polypeptides are those described as n to m herein. Inpreferred embodiments, the application is directed to nucleic acidmolecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequences encoding polypeptides having the amino acidsequence ofthe specific N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Polypeptide fragments of the present invention include polypeptidescomprising or alternatively, consisting of, an amino acid sequencecontained in SEQ ID NO:2, encoded by the cDNA contained in the depositedplasmid, or encoded by nucleic acids which hybridize (e.g., understringent hybridization conditions) to the nucleotide sequence containedin the deposited plasmid, or shown in FIGS. 1A–B (SEQ ID NO:1) or thecomplementary strand thereto. Protein fragments may be “free-standing,”or comprised within a larger polypeptide of which the fragment forms apart or region, most preferably as a single continuous region.Representative examples of polypeptide fragments of the invention,include, for example, fragments that comprise or alternatively, consistof from about amino acid residues: −25 to −1, 1 to 50, 51 to 100, 101 to130, 131 to 169, 170 to 191, and/or 192 to 203 of SEQ ID NO:2. Moreover,polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of T1R-like ligandII. Such fragments include amino acid residues that comprise alpha-helixand alpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) T1R-like ligand II(SEQ ID NO:2). Certain preferred regions are those set out in FIG. 3 andinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence depicted in FIGS. 1A–B(SEQ ID NO:2), such preferred regions include; Garnier-Robson predictedalpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasmanpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenbergalpha and beta amphipathic regions; Emini surface-forming regions; andJameson-Wolf high antigenic index regions, as predicted using thedefault parameters of these computer programs. Polynucleotides encodingthese polypeptides are also encompassed by the invention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of T1R-like ligand II. Preferred embodiments ofthe invention in this regard include fragments that comprise alpha-helixand alpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of T1R-likeligand II.

The data representing the structural or functional attributes ofT1R-like ligand II set forth in FIG. 3 and/or Table 2, as describedherein, was generated using the various modules and algorithms of theDNA*STAR set on default parameters. In a preferred embodiment, the datapresented in columns VIII, IX, XIII, and XIV of Table I can be used todetermine regions of T1R-like ligand II which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table 2, be represented or identified by using tabularrepresentations ofthe data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table 2). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table 2include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 1A–B.As set out in FIG. 3 and in Table 2, such preferred regions includeGamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

TABLE 2 Res Position I II III IV V VI VI VIII IX X XI XII XIII XIV Met 1A . . . . T . 0.16 −0.67 * . . 2.03 1.23 Gly 2 . . . . T T . 0.26 −0.41. . . 2.20 0.67 Asp 3 . . . . T T . −0.17 0.07 . . . 1.38 0.55 Lys 4 . .B . . T . 0.01 0.33 * * . 0.76 0.46 Ile 5 . . B . . . . −0.30 0.14 * * .0.34 0.72 Trp 6 . . B . . . . 0.09 0.50 * * . −0.18 0.37 Leu 7 . . B . .T . −0.42 0.93 * . . −0.20 0.29 Pro 8 . . B . . T . −1.23 1.57 * * .−0.20 0.31 Phe 9 . . B . . T . −2.09 1.57 . * . −0.20 0.24 Pro 10 . . B. . T . −2.01 1.34 . * . −0.20 0.24 Val 11 . A B . . . . −2.31 1.34 . .. −0.60 0.13 Leu 12 . A B . . . . −2.09 1.41 . . . −0.60 0.15 Leu 13 . AB . . . . −2.69 1.13 . . . −0.60 0.10 Leu 14 . A B . . . . −2.20 1.39 .. . −0.60 0.11 Ala 15 A A . . . . . −2.20 1.17 . . . −0.60 0.20 Ala 16 .A B . . . . −2.20 0.91 . . . −0.60 0.38 Leu 17 . A B . . . . −2.20 0.87. . . −0.60 0.34 Pro 18 . A B . . . . −2.20 0.87 . . . −0.60 0.28 Pro 19. . B . . . . −1.60 1.06 . . . −0.40 0.23 Val 20 . . B . . . . −1.360.99 . . . −0.40 0.43 Leu 21 . . B . . . . −1.36 0.73 . . . −0.40 0.27Leu 22 . . B . . T . −1.13 0.80 . . . −0.20 0.18 Pro 23 . . B . . T .−1.27 0.87 . . . −0.20 0.24 Gly 24 . . B . . T . −1.76 0.66 . . . −0.200.29 Ala 25 . . B . . T . −1.21 0.76 . . . −0.20 0.31 Ala 26 . . B B . .. −0.61 0.56 . . . −0.60 0.29 Gly 27 . . B B . . . −0.10 0.56 . . .−0.60 0.45 Phe 28 . . B B . . . −0.70 0.51 . * . −0.60 0.59 Thr 29 . . B. . T . −0.36 0.70 . * F 0.20 0.48 Pro 30 . . B . . T . −0.07 0.20 . * F0.75 0.82 Ser 31 . . B . . T . 0.52 0.16 . * F 1.15 1.27 Leu 32 . . . .. T C 0.17 −0.63 . * F 2.50 1.46 Asp 33 . . . . T T . 0.56 −0.33 . * F2.50 0.82 Ser 34 . . . . . T C 0.17 −0.27 . * F 2.05 0.88 Asp 35 . . . .T T . 0.07 0.13 . * F 1.40 0.93 Phe 36 . . B . . T . −0.44 −0.07 . * F1.35 0.80 Thr 37 . . B B . . . 0.16 0.61 . * . −0.35 0.49 Phe 38 . . B B. . . −0.43 0.66 . * . −0.60 0.46 Thr 39 . . B B . . . −0.48 1.16 . * .−0.26 0.53 Leu 40 . . . B . . C −0.48 0.80 . * . 0.28 0.37 Pro 41 . . .. . T C 0.27 0.71 . * . 1.02 0.73 Ala 42 . . . . T T . 0.58 −0.07 . . F2.76 1.01 Gly 43 . . . . T T . 0.61 −0.56 . . F 3.40 2.13 Gln 44 . . . .T T . 0.22 −0.67 . . F 2.91 0.74 Lys 45 . A . . T . . 0.79 −0.31 . . F1.87 0.63 Glu 46 . A B . . . . 1.00 −0.06 . . F 1.28 1.00 Cys 47 . A B .. . . 1.38 −0.09 . . . 0.79 1.00 Phe 48 . A B . . . . 1.12 −0.06 . . .0.30 0.77 Tyr 49 . A B . . . . 0.91 0.56 . . . −0.60 0.44 Gln 50 . . B .. . . 0.06 0.99 . . . −0.25 1.27 Pro 51 . . B . . . . 0.10 1.10 . * .−0.25 1.21 Met 52 A A . . . . . 0.18 0.31 . * . −0.15 1.55 Pro 53 A A .. . . . 0.58 0.06 . * . −0.30 0.90 Leu 54 A A . . . . . 0.01 0.04 . * .−0.30 0.78 Lys 55 A A . . . . . 0.01 0.30 . * . −0.30 0.65 Ala 56 A A .. . . . −0.67 −0.31 . * . 0.30 0.73 Ser 57 A A . . . . . −0.07 −0.06 . *. 0.30 0.62 Leu 58 A A . . . . . −0.10 −0.74 . * . 0.60 0.54 Glu 59 A A. . . . . 0.71 0.01 * * . −0.30 0.83 Ile 60 A A . . . . . −0.19−0.09 * * . 0.45 1.08 Glu 61 A A . . . . . −0.41 0.17 * * . −0.30 0.97Tyr 62 A A . . . . . −0.11 0.17 * * . −0.30 0.46 Gln 63 . A B . . . .0.36 0.17 * * . −0.15 1.10 Val 64 A A . . . . . −0.23 −0.09 * * . 0.300.63 Leu 65 A A . . . . . 0.31 0.41 . . . −0.60 0.41 Asp 66 A A . . . .. −0.50 0.09 . . F −0.15 0.23 Gly 67 A . . . . T . −0.26 0.37 * . F 0.250.26 Ala 68 A . . . . T . −1.14 −0.27 * * F 0.85 0.52 Gly 69 A . . . . T. −0.29 −0.27 . * . 0.70 0.22 Leu 70 A . . . . T . −0.18 −0.27 . * .0.70 0.37 Asp 71 A A . . . . . −0.21 0.09 . * . −0.30 0.32 Ile 72 . A B. . . . −0.68 0.09 . * . −0.30 0.44 Asp 73 . A B . . . . −0.68 0.34 . *. −0.30 0.44 Phe 74 . A B . . . . −0.63 0.16 . * . −0.30 0.26 His 75 A A. . . . . −0.03 0.54 . * . −0.60 0.50 Leu 76 A A . . . . . −0.03 0.29. * . 0.04 0.47 Ala 77 . A . . . . C 0.51 0.29 . * . 0.58 0.93 Ser 78 .. . . . T C 0.56 −0.07 . * F 2.07 0.68 Pro 79 . . . . . T C 0.94 −0.57 *. F 2.86 1.64 Glu 80 . . . . T T . 0.17 −0.77 * . F 3.40 2.35 Gly 81 A .. . . T . 0.12 −0.59 . . F 2.66 1.45 Lys 82 A A . . . . . 0.01 −0.33 . .F 1.47 0.69 Thr 83 A A . . . . . 0.31 0.03 . . F 0.53 0.35 Leu 84 A A .. . . . 0.52 0.03 . * . 0.04 0.61 Val 85 A A . . . . . 0.63 0.00 . . .−0.30 0.53 Phe 86 A A . . . . . 1.02 0.00 * . . −0.30 0.71 Glu 87 A A .. . . . 0.68 −0.49 . . F 0.94 1.73 Gln 88 A A . . . . . 0.99 −0.79 . . F1.58 3.13 Arg 89 A A . . . . . 1.46 −1.43 . . F 1.92 6.03 Lys 90 . . . .T T . 1.46 −1.79 . . F 3.06 3.44 Ser 91 . . . . T T . 2.12 −1.14 * . F3.40 1.48 Asp 92 . . . . T T . 1.81 −1.04 * . F 3.06 1.03 Gly 93 . . . .. T C 0.96 −0.56 * . F 2.37 0.74 Val 94 . . . B . . C 0.84 0.09 . . .0.58 0.41 His 95 . . B B . . . 0.49 −0.30 . . . 0.64 0.43 Thr 96 . . B B. . . 0.79 0.19 * * . −0.30 0.62 Val 97 . . B B . . . −0.07 −0.24 * . .0.45 1.45 Glu 98 . . B B . . . −0.07 −0.24 * . F 0.45 0.79 Thr 99 A . .. . . . 0.79 −0.31 * . F 0.65 0.54 Glu 100 A . . . . . . 0.58 −0.80 * .F 1.10 1.22 Val 101 A . . . . T . 0.29 −0.69 . . F 1.30 1.10 Gly 102 A .. . . T . 0.44 −0.07 . . F 0.85 0.76 Asp 103 A . . . . T . −0.22 0.23 .. . 0.10 0.38 Tyr 104 A . B . . T . −0.61 0.80 . . . −0.20 0.27 Met 105. . B . . . . −0.61 0.94 * . . −0.40 0.24 Phe 106 . . B . . . . 0.240.51 * . . −0.40 0.24 Cys 107 . . B . . . . 0.28 0.91 * * . −0.40 0.24Phe 108 . . B . . . . −0.42 0.64 * * . −0.40 0.36 Asp 109 . . . . T . .−0.48 0.81 . . F 0.15 0.36 Asn 110 . . . . T T . −0.19 0.41 * . F 0.350.89 Thr 111 . . . . . T C −0.38 0.33 * . F 0.60 1.49 Phe 112 . . . . .T C −0.01 0.23 * . F 0.45 0.62 Ser 113 . . . . . T C 0.69 0.61 * . F0.15 0.52 Thr 114 A . . B . . . 0.73 0.21 * . F −0.15 0.62 Ile 115 A . .B . . . −0.12 −0.27 * . F 0.60 1.44 Ser 116 A . . B . . . −0.70 −0.41 *. F 0.45 0.80 Glu 117 A . . B . . . −0.70 −0.11 * . F 0.45 0.39 Lys 118A . . B . . . −1.10 0.19 . . F −0.15 0.48 Val 119 A . . B . . . −0.790.29 . . . −0.30 0.31 Ile 120 A . . B . . . −0.71 −0.10 . . . 0.30 0.31Phe 121 A . . B . . . −1.30 0.59 . . . −0.60 0.13 Phe 122 A . . B . . .−2.11 1.27 . . . −0.60 0.12 Glu 123 A . . B . . . −2.16 1.31 . . . −0.600.14 Leu 124 A . . B . . . −1.30 0.63 * . . −0.60 0.27 Ile 125 A . . B .. . −1.01 0.24 * . . −0.30 0.51 Leu 126 A . . B . . . −0.66 0.07 * . .−0.30 0.29 Asp 127 A . . B . . . 0.04 0.50 * . . −0.60 0.35 Asn 128 A .. . . T . 0.04 −0.19 * . F 0.85 0.86 Met 129 A . . . . T . 0.27 −0.47 *. F 1.00 1.81 Gly 130 A . . . . T . 1.16 −0.66 * . F 1.30 1.09 Glu 131 A. . . . T . 1.97 −0.26 * . F 1.00 1.18 Gln 132 A A . . . . . 1.97−0.66 * . F 0.90 2.06 Ala 133 A A . . . . . 1.97 −0.87 * . F 0.90 3.61Gln 134 A A . . . . . 2.57 −1.30 . . F 0.90 3.61 Glu 135 A A . . . . .2.62 −1.30 . * F 0.90 3.48 Gln 136 A A . . . . . 2.67 −0.79 . . F 0.903.62 Glu 137 A A . . . . . 2.71 −1.29 . * F 0.90 4.18 Asp 138 A A . . .. . 3.06 −1.69 * * F 0.90 4.83 Trp 139 A A . . . . . 2.17 −0.93 * . F0.90 4.37 Lys 140 A A . . . . . 1.86 −0.64 * . F 0.90 1.77 Lys 141 . A .. T . . 1.51 −0.16 * * F 1.00 1.53 Tyr 142 . A . . T . . 1.20 0.27 * . .0.25 1.44 Ile 143 . . B . . . . 1.20 −0.16 . * F 0.97 1.04 Thr 144 . . B. . . . 0.60 −0.16 * . F 0.99 0.87 Gly 145 . . B . . T . −0.26 0.53 * .F 0.46 0.39 Thr 146 . . B . . T . −0.30 0.46 . . F 0.63 0.46 Asp 147 . .B . . T . −0.66 −0.23 . . F 1.70 0.53 Ile 148 A . . . . T . 0.28 −0.10. * . 1.38 0.53 Leu 149 A A . . . . . −0.22 −0.53 * * . 1.11 0.73 Asp150 A A . . . . . 0.12 −0.33 * * . 0.64 0.36 Met 151 A A . . . . . 0.43−0.33 * . . 0.47 0.89 Lys 152 A A . . . . . −0.46 −1.01 * * . 0.75 1.81Leu 153 A A . . . . . −0.38 −1.01 * * F 0.75 0.76 Glu 154 A A . . . . .0.43 −0.33 * * F 0.45 0.63 Asp 155 A A . . . . . 0.13 −0.94 * * F 0.750.55 Ile 156 A A . . . . . −0.16 −0.56 * * . 0.60 0.89 Leu 157 A A . . .. . −0.20 −0.56 * . . 0.60 0.36 Glu 158 A A . . . . . 0.31 −0.16 * . F0.45 0.35 Ser 159 A . . . . T . −0.58 0.23 * . F 0.25 0.66 Ile 160 A . .. . T . −0.53 0.23 * . F 0.25 0.56 Asn 161 A . . . . T . 0.06 −0.46 * *F 0.85 0.65 Ser 162 A . . . . T . 0.98 −0.07 * * F 0.85 0.65 Ile 163 A .. . . . . 0.17 −0.46 * * F 0.80 1.82 Lys 164 A . . . . . . 0.17−0.46 * * F 0.99 0.93 Ser 165 . . B . . . . 1.10 −0.47 * * F 1.33 0.93Arg 166 . . . . T . . 0.80 −0.86 * * F 2.52 2.66 Leu 167 . . B . . . .0.76 −1.16 * * F 2.46 1.78 Ser 168 . . . . T T . 1.61 −0.73 * * F 3.401.32 Lys 169 . . . . T T . 0.68 −0.61 . * F 2.91 0.91 Ser 170 . . . . .T C 0.98 0.07 * * F 1.47 0.78 Gly 171 . . . . T T . 0.56 −0.21 . * F2.08 1.00 His 172 . A B . . . . 0.56 −0.11 . . F 0.79 0.72 Ile 173 . A B. . . . 0.04 0.57 * * . −0.60 0.45 Gln 174 . A B . . . . 0.11 0.87 * * .−0.60 0.37 Thr 175 . A B . . . . −0.18 0.44 * * . −0.60 0.53 Leu 176 . AB . . . . −0.53 0.44 * * . −0.60 0.77 Leu 177 . A B . . . . −0.500.54 * * . −0.60 0.39 Arg 178 A A . . . . . −0.20 0.14 * * . −0.30 0.46Ala 179 A A . . . . . −0.09 0.16 * . . −0.30 0.57 Phe 180 A A . . . . .0.22 −0.53 * * . 0.75 1.35 Glu 181 A A . . . . . 1.14 −1.21 * * . 0.751.15 Ala 182 A A . . . . . 1.96 −1.21 * . . 0.75 2.22 Arg 183 A A . . .. . 0.96 −1.31 * . F 0.90 4.13 Asp 184 A . . . . T . 1.54 −1.41 * * F1.30 1.67 Arg 185 A . . . . T . 2.24 −1.01 * * F 1.30 2.87 Asn 186 A . .. . T . 1.94 −1.51 . * F 1.30 2.54 Ile 187 A . . . . T . 2.53 −1.13 . .F 1.64 2.03 Gln 188 . . . . . . C 1.72 −0.73 . . F 1.98 1.67 Glu 189 . .B . . . . 1.72 0.06 . . F 1.07 0.90 Ser 190 . . . . . . C 1.72 −0.34 * .F 2.36 2.14 Asn 191 . . . . T T . 0.87 −1.03 * . F 3.40 2.42 Phe 192 . .. . T T . 1.76 −0.79 . * F 3.06 1.04 Asp 193 . . . . T T . 1.06 −0.39 .. F 2.42 1.25 Arg 194 . . . . T T . 0.77 0.01 . . . 1.18 0.67 Val 195 .. . B T . . 0.77 0.53 . . . 0.14 0.81 Asn 196 . . . B T . . 0.17 0.13 .. . 0.10 0.65 Phe 197 . . . B T . . 0.01 0.74 . * . −0.20 0.33 Trp 198 A. . B . . . 0.01 1.39 . * . −0.60 0.33 Ser 199 A . . B . . . −0.91 1.14. * . −0.60 0.33 Met 200 A . . B . . . −0.91 1.43 * * . −0.60 0.31 Val201 A . . B . . . −1.77 1.29 * . . −0.60 0.22 Asn 202 . . B B . . .−1.67 1.01 * . . −0.60 0.12 Leu 203 A . . B . . . −2.23 1.24 * . . −0.600.12 Val 204 . . B B . . . −2.79 1.27 . . . −0.60 0.12 Val 205 . . B B .. . −3.04 1.27 . . . −0.60 0.06 Met 206 . . B B . . . −2.49 1.51 * * .−0.60 0.05 Val 207 . . B B . . . −3.08 1.21 . . . −0.60 0.09 Val 208 . .B B . . . −3.16 1.07 * . . −0.60 0.13 Val 209 A . . B . . . −2.30 1.11 *. . −0.60 0.09 Ser 210 A . . B . . . −2.30 0.90 . . . −0.60 0.21 Ala 211A . . B . . . −1.94 0.90 . . . −0.60 0.21 Ile 212 A . . B . . . −1.691.01 . . . −0.60 0.44 Gln 213 A . . B . . . −1.64 0.99 . . . −0.60 0.32Val 214 A . . B . . . −0.74 1.29 . . . −0.60 0.26 Tyr 215 . . B B . . .−0.74 0.79 . . . −0.60 0.75 Met 216 . . B B . . . −0.97 0.49 . . . −0.600.58 Leu 217 A . . B . . . −0.78 0.77 * * . −0.60 0.65 Lys 218 A . . B .. . −0.78 0.91 * . . −0.60 0.36 Ser 219 A A . . . . . 0.08 0.16 * . .−0.30 0.63 Leu 220 A A . . . . . 0.37 −0.46 * . . 0.45 1.27 Phe 221 A A. . . . . 1.08 −1.14 * . . 0.75 1.27 Glu 222 A A . . . . . 1.93 −1.14 *. F 0.90 1.85 Asp 223 A A . . . . . 1.59 −1.53 * . F 1.21 4.50 Lys 224 AA . . . . . 2.00 −1.83 . . F 1.52 6.96 Arg 225 A . . . . T . 2.50 −2.61. . F 2.23 7.87 Lys 226 A . . . . T . 2.81 −2.13 . * F 2.54 6.80 Ser 227. . . . T T . 2.42 −1.70 * . . 3.10 4.35 Arg 228 A . . . T T . 2.03−1.27 * . . 2.79 2.84 Thr 229 . . B . . . . 1.60 −0.84 * . . 1.88 1.81

Among highly preferred fragments in this regard are those that compriseregions of T1R-like ligand II that combine several structural features,such as several of the features set out above.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail herein, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting T1R-like ligand II expressionas described herein or as agonists and antagonists capable of enhancingor inhibiting T1R-like ligand II protein function. Further, suchpolypeptides can be used in the yeast two-hybrid system to “capture”T1R-like ligand II binding proteins which are also candidate agonist andantagonist according to the present invention. The yeast two hybridsystem is described in Fields and Song, Nature 340:245–246 (1989).

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO: 2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. 97655 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO: 1 or contained in ATCC depositNo. 97655 under stringent hybridization conditions or lower stringencyhybridization conditions as defined herein. The present inventionfurther encompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:2), polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined herein.

The term “epitopes, ” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope, ” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described herein. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998–4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross- reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al. (1984), supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art.

For instance, the immunologically important epitope in the coat proteinof foot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁–C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G. et al., Science219:660–666 (1983). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals.

Peptides that are extremely hydrophobic and those of six or fewerresidues generally are ineffective at inducing antibodies that bind tothe mimicked protein; longer, soluble peptides, especially thosecontaining proline residues, usually are effective. Sutcliffe et al.,supra, at 661. For instance, 18 of 20 peptides designed according tothese guidelines, containing 8–39 residues covering 75% of the sequenceof the influenza virus hemagglutinin HA1 polypeptide chain, inducedantibodies that reacted with the HA1 protein or intact virus; and 12/12peptides from the MuLV polymerase and 18/18 from the rabies glycoproteininduced antibodies that precipitated the respective proteins.

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 890, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767–778 (1984); Sutcliffe et al., Science 219:660–666 (1983)).

Non-limiting examples of antigenic polypeptides that can be used togenerate T1R-like ligand II specific antibodies or fragments, includethe following: a polypeptide comprising amino acid residues from about17 to about 26 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 56 to about 72 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 103 to about 120 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 136 toabout 149 in SEQ ID NO:2; and a polypeptide comprising amino acidresidues from about 155 to about 171 in SEQ ID NO:2. As indicated above,the inventors have determined that the above polypeptide fragments areantigenic regions of the T1R-like II protein.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes posttranslationprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance,Wilson, I. A. et al., Cell 37:767–778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle et al., J. Gen. Virol.66:2347–2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131–5135(1985), further described in U.S. Pat. No. 4,631,211). For instance, ashort epitope-bearing amino acid sequence may be fused to a largerpolypeptide which acts as a carrier during recombinant production andpurification, as well as during immunization to produce anti-peptideantibodies. Epitope-bearing peptides also may be synthesized using knownmethods of chemical synthesis.

For instance, Houghten has described a simple method for synthesis oflarge numbers of peptides, such as 10–20 mg of 248 different 13 residuepeptides representing single amino acid variants of a segment of the HAIpolypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. , Proc. Natl. Acad.Sci. USA 82:5131–5135 (1985). This “Simultaneous Multiple PeptideSynthesis (SMPS)” process is further described in U.S. Pat. No.4,631,211 to Houghten et al. (1986). In this procedure the individualresins for the solid-phase synthesis of various peptides are containedin separate solvent-permeable packets, enabling the optimal use of themany identical repetitive steps involved in solid-phase methods. Acompletely manual procedure allows 500–1000 or more syntheses to beconducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347–2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid.

For instance, peptides containing cysteine residues may be coupled to acarrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimideester (MBS), while other peptides may be coupled to carriers using amore general linking agent such as glutaraldehyde. Animals such asrabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermalinjection of emulsions containing about 100 μg of peptide or carrierprotein and Freund's adjuvant or any other adjuvant known forstimulating an immune response. Several booster injections may beneeded, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody which can be detected, forexample, by ELISA assay using free peptide adsorbed to a solid surface.The titer of anti-peptide antibodies in serum from an immunized animalmay be increased by selection of anti-peptide antibodies, for instance,by adsorption to the peptide on a solid support and elution of theselected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, T1R-like ligand IIpolypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions ofthe heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84–86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric T1R-like ligand IIprotein or protein fragment alone (Fountoulakis et al., J. Biochem.270:3958–3964 (1995)).

The invention further provides for the proteins containing T1R-likeligand II polypeptide sequences encoded by the polynucleotides of theinvention.

The T1R-like ligand II polypeptides of the invention may be in monomersor multimers (i.e., dimers, trimers, tetramers, and higher multimers).Accordingly, the present invention relates to monomers and multimers ofthe T1R-like ligand II proteins of the invention, their preparation, andcompositions (preferably, pharmaceutical compositions) containing them.In specific embodiments, the polypeptides ofthe invention are monomers,dimers, trimers or tetramers. In additional embodiments, the multimersof the invention are at least dimers, at least trimers, or at leasttetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyT1R-like ligand II proteins of the invention (including T1R-like ligandII fragments, variants, and fusion proteins, as described herein). Thesehomomers may contain T1R-like ligand II proteins having identical ordifferent polypeptide sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only T1R-like ligand II proteinshaving an identical polypeptide sequence. In another specificembodiment, a homomer of the invention is a multimer containing T1R-likeligand II proteins having different polypeptide sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing T1R-like ligand II proteins having identical or differentpolypeptide sequences) or a homotrimer (e.g., containing T1R-like ligandII proteins having identical or different polypeptide sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing only polypeptidesequences that do not correspond to a polypeptide sequences encoded bythe T1R-like ligand II gene) in addition to the T1R-like ligand IIproteins of the invention. In a specific embodiment, the multimer of theinvention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the heteromeric multimer of the invention is atleast a heterodimer, at least a heterotrimer, or at least aheterotetramer.

Multimers ofthe invention maybe the result of hydrophobic, hydrophilic,ionic and/or covalent associations and/or may be indirectly linked, byfor example, liposome formation. Thus, in one embodiment, multimers ofthe invention, such as, for example, homodimers or homotrimers, areformed when proteins of the invention contact one another in solution.In another embodiment, heteromultimers of the invention, such as, forexample, heterotrimers or heterotetramers, are formed when proteins ofthe invention contact antibodies to the polypeptides of the invention(including antibodies to the heterologous polypeptide sequence in afusion protein of the invention) in solution. In other embodiments,multimers of the invention are formed by covalent associations withand/or between the T1R-like ligand II proteins of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence of the protein (e.g., thepolypeptide sequence recited in SEQ ID NO:2 or the polypeptide encodedby the deposited cDNA plasmid). In one instance, the covalentassociations are cross-linking between cysteine residues located withinthe polypeptide sequences of the proteins which interact in the native(i.e., naturally occurring) polypeptide. In another instance, thecovalent associations are the consequence of chemical or recombinantmanipulation. Alternatively, such covalent associations may involve oneor more amino acid residues contained in the heterologous polypeptidesequence in a T1R-like ligand II fusion protein. In one example,covalent associations are between the heterologous sequence contained ina fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925).In a specific example, the covalent associations are between theheterologous sequence contained in a T1R-like ligand II-Fc fusionprotein of the invention (as described herein). In another specificexample, covalent associations of fusion proteins of the invention arebetween heterologous polypeptde sequences from another protein that iscapable of forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication No. WO 98/49305,the contents of which are herein incorporated by reference in itsentirety).

In another embodiment, two or more T1R-like ligand II polypeptides oftheinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple T1R-like ligandII polypeptides separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer T1R-like ligand II polypeptides ofthe invention involves use of T1R-like ligand II polypeptides fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric T1R-like ligand II proteins are those described inInternational application publication nunmper WO 94/10308, herebyincorporated by reference. Recombinant fusion proteins comprising asoluble T1R-like ligand II polypeptide fused to a peptide that dimerizesor trimerizes in solution are expressed in suitable host cells, and theresulting soluble multimeric T1R-like ligand II is recovered from theculture supernatant using techniques known in the art.

In another example, proteins of the invention are associated byinteractions between Flag(r) polypeptide sequence contained inFlag(r)-T1R-like ligand II fusion proteins of the invention. In afurther embodiment, associations proteins ofthe invention are associatedby interactions between heterologous polypeptide sequence contained inFlag(r)-T1R-like ligand II fusion proteins of the invention andanti-Flag(r) antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, proteins desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the polypeptidesequence of the proteins desired to be contained in the multimer (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety).

Further, proteins of the invention may be routinely modified by theaddition of cysteine or biotin to the C terminus or N-terminus of thepolypeptide sequence of the protein and techniques known in the art maybe applied to generate multimers containing one or more of thesemodified proteins (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing theprotein components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers ofthe invention maybe generated using geneticengineering techniques known in the art. In one embodiment, proteinscontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain and which can beincorporated by membrane reconstitution techniques into liposomes (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety).

The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

Fusion Polypeptides

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting ofthe first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84–86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958–3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972–897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins ofthe invention maybe generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as modulating the activity of T1R-like ligandII agonists and antagonists of the polypeptides. See, generally, U.S.Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, andPatten et al., Curr. Opinion Biotechnol. 8:724–33 (1997); Harayama,Trends Biotechnol. 16(2):76–82 (1998); Hansson, et al., J. Mol. Biol.287:265–76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308–13(1998) (each of these patents and publications are hereby incorporatedby reference in its entirety). In one embodiment, alteration ofpolynucleotides corresponding to SEQ ID NO:1 and the polypeptidesencoded by these polynucleotides may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments into adesired T1R-like ligand II molecule by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are members of the IL-1R family, for example IL-1RI, IL-1RII,and sIL-1RII.

Transgenic Animals

The proteins of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., nucleic acids of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691–698 (1994); Carver et al., Biotechnology(NY) 11:1263–1270 (1993); Wright et al., Biotechnology (NY) 9:830–834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148–6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313–321(1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol.3:1803–1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717–723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171–229 (1989), which is incorporated by referenceherein in its entirety. Further, the contents of each ofthe documentsrecited in this paragraph is herein incorporated by reference in itsentirety.

Further techniques known in the art may be used to introduce thetransgene (i.e., nucleic acids of the invention) into animals include,for example, those techniques described in U.S. Pat. No. 5,464,764(Capecchi et al., Positive-Negative Selection Methods and Vectors); U.S.Pat. No. 5,631,153 (Capecchi et al., Cells and Non-Human OrganismsContaining Predetermined Genomic Modifications and Positive-NegativeSelection Methods and Vectors for Making Same); U.S. Pat. No. 4,736,866(Leder et al., Transgenic Non-Human Animals); and U.S. Pat. No.4,873,191 (Wagner et al., Genetic Transformation of Zygotes); each ofwhich is hereby incorporated by reference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64–66 (1996); Wilmut et al., Nature 385:810–813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl.Acad. Sci. USA 89:6232–6236 (1992)). The regulatory sequences requiredfor such a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart. When it is desired that the polynucleotide transgene be integratedinto the chromosomal site of the endogenous gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al., Science265:103–106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph is hereinincorporated by reference in its entirety.

Once transgenic animals have been generated, the expression oftherecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of T1R-like ligand II polypeptides,studying conditions and/or disorders associated with aberrant T1R-likeligand II expression, and in screening for compounds effective inameliorating such conditions and/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the proteins of the invention, or alternatively,that are genetically engineered not to express the proteins of theinvention (e.g., knockouts) are administered to a patient in vivo. Suchcells may be obtained from the patient (i.e., animal, including human)or an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. (See, for example, Anderson et al. U.S. Pat. No.5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of whichis incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

T1R-like Ligand II Antibodies and Antibody Therapy

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion ofthe following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described herein and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60–69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547–1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide ofthe presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or K_(d) less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10 ⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed herein). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981–1988 (1998); Chenet al., Cancer Res. 58(16):3668–3678 (1998); Harrop et al., J. Immunol.161(4):1786–1794 (1998); Zhu et al., Cancer Res. 58(15):3209–3214(1998); Yoon et al., J. Immunol. 160(7):3170–3179(1998); Prat et al., J.Cell. Sci. 111(Pt2):237–247 (1998); Pitard et al., J. Immunol. Methods205(2):177–190 (1997); Liautard et al., Cytokine 9(4):233–241 (1997);Carlson et al., J. Biol. Chem. 272(17):11295–11301 (1997); Taryman etal., Neuron 14(4):755–762 (1995); Muller et al., Structure6(9):1153–1167 (1998); Bartunek et al., Cytokine 8(1):14–20 (1996)(whichare all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail herein, the antibodies ofthe presentinvention maybe used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art.For example, a polypeptide of the invention can be administered tovarious host animals including, but not limited to, rabbits, mice, rats,etc. to induce the production of sera containing polyclonal antibodiesspecific for the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563–681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 16). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein.

Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41–50 (1995); Ames et al., J. Immunol.Methods 184:177–186 (1995); Kettleborough et al., Eur. J. Immunol.24:952–958 (1994); Persic et al., Gene 187 9–18 (1997); Burton et al.,Advances in Immunology 57:191–280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail herein. For example, techniquesto recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864–869(1992); and Sawai et al., AJRI 34:26–34 (1995); and Better et al.,Science 240:1041–1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46–88 (1991); Shu etal., PNAS 90:7995–7999 (1993); and Skerra et al., Science 240:1038–1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191–202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety.

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand a framework regions from a human immunoglobulin molecule. Often,framework residues in the human framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Antibodies can be humanized using a variety of techniques known in theart including, for example, CR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489–498 (1991); Studnicka et al., Protein Engineering7(6):805–814 (1994); Roguska. et al., PNAS 91:969–973 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention.

Monoclonal antibodies directed against the antigen can be obtained fromthe immunized, transgenic mice using conventional hybridoma technology.The human immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65–93 (1995).For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047;WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899–903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437–444;(1989) and Nissinoff, J. Immunol. 147(8):2429–2438 (1991)). For exampantibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedherein, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification ofthe ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence oftheantibody is determined, the nucleotide sequence of the antibody may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed herein. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457–479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed herein, one or more amino acid substitutionsmay be made within the framework regions, and, preferably, the aminoacid substitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851–855 (1984);Neuberger et al., Nature 312:604–608 (1984); Takeda et al., Nature314:452–454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423–42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879–5883 (1988); and Wardet al., Nature 334:544–54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038–1041 (1988)).

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression ofthe entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed herein.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., met allothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

Preferably, bacterial cells such as Escherichia coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101–3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503–5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systemsmaybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non- essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts. (e.g., see Logan& Shenk, Proc. Natl. Acad. Sci. USA 81:355–359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51–544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1–2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488–505; Wu and Wu, Biotherapy 3:87–95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573–596 (1993); Mulligan,Science 260:926–932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191–217 (1993); May, 1993, TIB TECH 11(5):155–215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) ofthe presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides ofthepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., herein, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91–99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428–1432 (1992); Fell et al., J. Immunol.146:2446–2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Feregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fe portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fe portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535–10539 (1991); Zheng et al., J. Immunol.154:5590–5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337–11341(1992) (said references incorporated by reference in theirentireties).

As discussed, herein, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2 may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:2 maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84–86 (1988). The polypeptides ofthe present invention fusedorconjugatedto an antibodyhaving disulfide- linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958–3964(1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52–58 (1995); Johanson et al., J. Biol. Chem.270:9459–9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic met al ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for met al ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive met al ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II)(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerlydaunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)), andanti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, á-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567–1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623–53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475–506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303–16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119–58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneofthe present invention maybe useful as a cell specific marker, or morespecifically as a cellular marker that is differentially expressed atvarious stages of differentiation and/or maturation of particular celltypes. Monoclonal antibodies directed against a specific epitope, orcombination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737–49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly herein (but are notintended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1–4 hours) at 4ø C, adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4ø C, washing the beads in lysis buffer and resuspendingthe beads in SDS/sample buffer. The ability of the antibody of interestto immunoprecipitate a particular antigen can be assessed by, e.g.,western blot analysis. One of skill in the art would be knowledgeable asto the parameters that can be modified to increase the binding of theantibody to an antigen and decrease the background (e.g., pre-clearingthe cell lysate with sepharose beads). For further discussion regardingimmunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%–20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity ofthe antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail herein. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, ofthepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or K_(d) less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Gene Therapy

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488–505 (1993); Wu and Wu, Biotherapy 3:87–95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573–596 (1993);Mulligan, Science 260:926–932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191–217 (1993); May, TIBTECH 11(5):155–215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding a T1R-like ligand II polypeptide, antibody, antagonist,agonist, or fragment or variant thereof, said nucleic acid sequencesbeing part of expression vectors that express the T1R-like ligand IIpolypeptide, a polypeptide fragment, antibody, antagonist, agonist, orvariant thereof in a suitable host. In particular, such nucleic acidsequences have promoters operably linked to the coding region, saidpromoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequences and any other desiredsequences are flanked by regions that promote homologous recombinationat a desired site in the genome, thus providing for intrachromosomalexpression of the encoding nucleic acids (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932–8935 (1989); Zijlstra et al., Nature342:435–438 (1989). In specific embodiments, the expressed antibodymolecule is a single chain antibody; alternatively, the nucleic acidsequences include sequences encoding both the heavy and light chains, orfragments thereof, of the antibody. As mentioned previously,polypeptides, polypeptide fragments, antagonists, agonists, and variantsthereof may also be expressed.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429–4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc.

In another embodiment, nucleic acid-ligand complexes can be formed inwhich the ligand comprises a fusogenic viral peptide to disruptendosomes, allowing the nucleic acid to avoid lysosomal degradation. Inyet another embodiment, the nucleic acid can be targeted in vivo forcell specific uptake and expression, by targeting a specific receptor(see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO 92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932–8935 (1989); Zijlstra et al., Nature342:435–438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding a polypeptide of the invention are used. For example,a retroviral vector can be used (see Miller et al., Meth. Enzymol.217:581–599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding thepolypeptide to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291–302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644–651 (1994); Kiem et al., Blood83:1467–1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129–141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110–114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499–503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3–10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431–434 (1991); Rosenfeld et al., Cell 68:143–155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225–234(1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775–783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289–300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599–618 (1993); Cohen et al., Meth. Enzymol. 217:618–644 (1993);Cline, Pharmac. Ther. 29:69–92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc. Particularly preferred are CD34+ cells.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences are introduced into the cells such that they areexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson,Cell 71:973–985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); andPittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)). Particularlypreferred are CD 34+ cells.

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Antisense and Ribozyme Antagonists

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in Ti R-likeligand II, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited plasmid ATCC Deposit No. 97655. Inone embodiment, antisense sequence is generated internally by theorganism, in another embodiment, the antisense sequence is separatelyadministered (see, for example, O'Connor, J., Neurochem. 56:560 (1991),and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance, Lee etal., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1300(1991). The methodsarebased onbinding of a polynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the T1R-like ligand II antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector or a portion thereof, is transcribed,producing an antisense nucleic acid (RNA) of the invention. Such avector would contain a sequence encoding the T1R-like ligand IIantisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others know in the art, used for replication andexpression in vertebrate cells. Expression of the sequence encodingT1R-like ligand II, or fragments thereof, can be by any promoter knownin the art to act in vertebrate, preferably human cells. Such promoterscan be inducible or constitutive. Such promoters include, but are notlimited to, the SV40 early promoter region (Bemoist and Chambon, Nature29:304–310 (1981), the promoter contained in the 3′ long terminal repeatof Rous sarcoma virus (Yamamoto et al., Cell 22:787–797 (1980), theherpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1441–1445 (1981), the regulatory sequences of the metallothioneingene (Brinster, et al., Nature 296:39–42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a T1R-likeligand II gene. However, absolute complementarity, although preferred,is not required. A sequence “complementary to at least a portion of anRNA,” referred to herein, means a sequence having sufficientcomplementarity to be able to hybridize with the RNA, forming a stableduplex; in the case of double stranded T1R-like ligand II antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid Generally, the larger the hybridizing nucleic acid, themore base mismatches with a T1R-like ligand II RNA it may contain andstill form a stable duplex (or triplex as the case may be). One skilledin the art can ascertain a tolerable degree of mismatch by use ofstandard procedures to determine the melting point of the hybridizedcomplex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333–335. Thus, oligonucleotides complementary to either the 5′- or3′- non-translated, non-coding regions of the T1R-like ligand II shownin FIGS. 1A–B could be used in an antisense approach to inhibittranslation of endogenous T1R-like ligand II mRNA. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are less efficient inhibitors oftranslation but could be used in accordance with the invention. Whetherdesigned to hybridize to the 5′-, 3′- or coding region of T1R-likeligand II mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553–6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648–652(1987); PCT Publication No. WO88/09810) or the blood-brain barrier (see,e.g., PCT Publication No. WO89/10134), hybridization-triggered cleavageagents. (See, e.g., Krol et al., BioTechniques 6:958–976 (1988)) orintercalating agents. (See, e.g., Zon, Pharm. Res. 5:539–549 (1988)). Tothis end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from′ the groupincluding, but not limited to, a phosphorothioate, a phosphorodithioate,a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625–6641 (1987)). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131–6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327–330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448–7451 (1988)), etc.

While antisense nucleotides complementary to the T1R-like ligand IIcoding region sequence could be used, those complementary to thetranscribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364; Sarver et al, Science 247:1222–1225 (1990). While ribozymesthat cleave mRNA at site specific recognition sequences can be used todestroy T1R-like ligand II mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature 334:585–591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within thenucleotide sequence of T1R-like ligand II (FIGS. 1A–B (SEQ ID NO: 1)).Preferably, the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the T1R-like ligand II mRNA; i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express T1R-likeligand II in vivo. DNA constructs encoding the ribozyme may beintroduced into the cell in the same manner as described above for theintroduction of antisense encoding DNA. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive promoter, such as, for example, pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous T1R-like ligand IImessages and inhibit translation. Since ribozymes unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the T1R-like ligand II gene and/or its promoter usingtargeted homologous recombination. (E.g., see Smithies et al., Nature317:230–234 (1985); Thomas & Capecchi, Cell 51:503–512 (1987); Thompsonet al., Cell 5:313–321 (1989); each of which is incorporated byreference herein in its entirety). For example, a mutant, non-functionalpolynucleotide of the invention (or a completely unrelated DNA sequence)flanked by DNA homologous to the endogenous polynucleotide sequence(either the coding regions or regulatory regions of the gene) can beused, with or without a selectable marker and/or a negative selectablemarker, to transfect cells that express polypeptides of the invention invivo. In another embodiment, techniques known in the art are used togenerate knockouts in cells that contain, but do not express the gene ofinterest. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the targeted gene. Suchapproaches are particularly suited in research and agricultural fieldswhere modifications to embryonic stem cells can be used to generateanimal offspring with an inactive targeted gene (e.g., see Thomas &Capecchi 1987 and Thompson 1989, supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art. The contents of each of the documents recited in thisparagraph is herein incorporated by reference in its entirety.

In other embodiments, antagonists according to the present inventioninclude soluble forms of T1R-like ligand II (e.g., fragments of theT1R-like ligand II shown in FIG. 2 (SEQ ID NO: 2) that include theligand binding domain from the extracellular region of the full lengthreceptor). Such soluble forms of the T1R-like ligand II, which may benaturally occurring or synthetic, antagonize T1R-like ligand II mediatedsignaling by competing with the cell surface bound forms of the receptorfor binding to T1R-like ligand II ligands. Antagonists of the presentinvention also include T1R-like ligand II Fc fusion proteins.

Antibodies according to the present invention may be prepared by any ofa variety of standard methods using T1R-like ligand II receptorimmunogens of the present invention. Such T1R-like ligand II receptorimmunogens include the T1R-like ligand II protein shown in FIGS. 1A–B(SEQ ID NO:2) (which may or may not include a leader sequence) andpolypeptide fragments of the receptor comprising the ligand binding,extracellular, transmembrane, the intracellular domains of T1R-likeligand II, or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists according tothe present invention can be raised according to the methods disclosedherein and and/or known in the art, such as, for example, those methodsdescribed in Tartaglia and Goeddel, J. Biol. Chem.267(7):4304–4307(1992)); Tartaglia et al., Cell 73:213–216 (1993)), andPCT Application WO 94/09137 (the contents of each of these threeapplications are herein incorporated by reference in their entireties),and are preferably specific to polypeptides of the invention having theamino acid sequence of SEQ ID NO: 2.

T1R-like Ligand II Related Disorder Diagnosis

For T1R-like ligand II related disorders, it is believed thatsubstantially altered (increased or decreased) levels of T1R-like ligandII gene expression can be detected in tissue or other cells or bodilyfluids (e.g., sera, plasma, urine, synovial fluid or spinal fluid) takenfrom an individual having such a disorder, relative to a “standard”T1R-like ligand II gene expression level, that is, the T1R-like ligandII gene expression level in tissue or bodily fluids from an individualnot having the disorder. Thus, the invention provides a diagnosticmethod useful during diagnosis of an T1R-like ligand II-relateddisorder, which involves measuring the expression level of the geneencoding the T1R-like ligand II in tissue or other cells or body fluidfrom an individual and comparing the measured gene expression level witha standard T1R-like ligand II gene expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of an T1R-like ligand II related disorder.

T1R-like ligand II-related disorders are believed to include, but arenot limited to, leukemia, lymphoma, arteriosclerosis, autoimmunediseases, inflammatory diseases, Alzheimer's disease, ophthalmicdiseases, apoptosis, intrauterine growth retardation, preeclampsia,pemphigus and psoriasis.

By individual is intended mammalian individuals, preferably humans. By“measuring the expression level of the gene encoding the T1R-like ligandII” is intended qualitatively or quantitatively measuring or estimatingthe level of the T1R-like ligand II protein or the level of the mRNAencoding the T1R-like ligand II protein in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to the T1R-likeligand II protein level or mRNA level in a second biological sample).Preferably, the T1R-like ligand II protein level or mRNA level in thefirst biological sample is measured or estimated and compared to astandard T1R-like ligand II protein level or mRNA level, the standardbeing taken from a second biological sample obtained from an individualnot having the disorder or being determined by averaging levels from apopulation of individuals not having the disorder. As will beappreciated in the art, once a standard T1R-like ligand II protein levelor mRNA level is known, it can be used repeatedly as a standard forcomparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains T1R-like ligand II protein or mRNA. As indicated,biological samples include body fluids (such as sera, plasma, urine,synovial fluid and spinal fluid) which contain secreted mature T1R-likeligand II, or tissue sources found to express T1R-like ligand IIprotein. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156–159 (1987). Levels ofmRNA encoding an T1R-like ligand II are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al,Cell 63:303–312 (1990). Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. T1R-like ligand II cDNAlabeled according to any appropriate method (such as the ³²P-multiprimedDNA labeling system (Amersham)) is used as probe. After hybridizationovernight, the filter is washed and exposed to x-ray film. cDNA for useas probe according to the present invention is described in the sectionsabove and will preferably at least 15 bp in length.

S1 mapping can be performed as described in Fujita et al., Cell49:357–367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe T1R-like ligand II). Northern blot analysis can be performed asdescribed above.

Preferably, levels of mRNA encoding the T1R-like ligand II are assayedusing the RT-PCR method described in Makino et al., Technique 2:295–301(1990). By this method, the radioactivities of the “amplicons” in thepolyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the T1R-likeligand II) is quantified using an imaging analyzer. RT and PCR reactioningredients and conditions, reagent and gel concentrations, and labelingmethods are well known in the art. Variations on the RT-PCR method willbe apparent to the skilled artisan.

Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

Assaying T1R-like ligand II levels in a biological sample can occurusing any art-known method. Preferred for assaying T1R-like ligand IIlevels in a biological sample are antibody-based techniques. Forexample, T1R-like ligand II expression in tissues can be studied withclassical immunohistological methods. In these, the specific recognitionis provided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of T1R-like ligand II for Western-blot or dot/slotassay (Jalkanen, M., et al., J. Cell. Biol. 101:976–985 (1985);Jalkanen, M., et al., J. Cell. Biol. 105:3087–3096 (1987)).

In this technique, which is based on the use of cationic solid phases,quantitation of T1R-like ligand II can be accomplished using isolatedT1R-like ligand II as a standard. This technique can also be applied tobody fluids. With these samples, a molar concentration of T1R-likeligand II will aid to set standard values of T1R-like ligand II contentfor different body fluids, like serum, plasma, urine, synovial fluid,spinal fluid, etc. The normal appearance of T1R-like ligand II amountscan then be set using values from healthy individuals, which can becompared to those obtained from a test subject.

Other antibody-based methods useful for detecting T1R-like ligand IIlevels include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). For example, T1R-likeligand II-specific monoclonal antibodies can be used both as animmunoadsorbent and as an enzyme-labeled probe to detect and quantifythe T1R-like ligand II. The amount of T1R-like ligand II present in thesample can be calculated by reference to the amount present in astandard preparation using a linear regression computer algorithm. Suchan ELISA for detecting a tumor antigen is described in Iacobelli et al.,Breast Cancer Research and Treatment 11:19–30 (1988). In another ELISAassay, two distinct specific monoclonal antibodies can be used to detectT1R-like ligand II in a body fluid. In this assay, one of the antibodiesis used as the immunoadsorbent and the other as the enzyme-labeledprobe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting T1R-likeligand II with immobilized antibody and, without washing, contacting themixture with the labeled antibody. The “two-step” assay involves washingbefore contacting the mixture with the labeled antibody. Otherconventional methods may also be employed as suitable. It is usuallydesirable to immobilize one component of the assay system on a support,thereby allowing other components of the system to be brought intocontact with the component and readily removed from the sample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

In addition to assaying T1R-like ligand II levels in a biological sampleobtained from an individual, T1R-like ligand II can also be detected invivo by imaging. Antibody labels or markers for in vivo imaging ofT1R-like ligand II include those detectable by X-radiography, NMR orESR. For X-radiography, suitable labels include radioisotopes such asbarium or cesium, which emit detectable radiation but are not overtlyharmful to the subject. Suitable markers for NMR and ESR include thosewith a detectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A T1R-like ligand II-specific antibody or antibody fragment which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a disorder. Itwill be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moietiesneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of ^(99m)Tc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain T1R-like ligand II. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, Burchiel, S. W. andRhodes, B. A. eds., Masson Publishing Inc., (1982)).

T1R-like ligand II specific antibodies for use in the present inventioncan be raised against the intact T1R-like ligand II or an antigenicpolypeptide fragment thereof, which may presented together with acarrier protein, such as an albumin, to an animal system (such as rabbitor mouse) or, if it is long enough (at least about 25 amino acids),without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to T1R-like ligand II. Fab and F(ab′)₂ fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316–325 (1983)). Thus, theseportions are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the T1R-like ligand IIor an antigenic fragment thereof can be administered to an animal inorder to induce the production of sera containing polyclonal antibodies.In a preferred method, a preparation of T1R-like ligand II protein isprepared and purified as described above to render it substantially freeof natural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or T1R-like ligand II binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology (Colligan, Current Protocols in Immunology, WileyInterscience, New York (1990–1996); Harlow & Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1988), Chapters 6–9, Current Protocols in Molecular Biology, Ausubel,infra, Chapter 11, entirely incorporated herein by reference). Ingeneral, such procedures involve immunizing an animal (preferably amouse) with an T1R-like ligand II antigen or, more preferably, with anT1R-like ligand II-expressing cell. Suitable cells can be recognized bytheir capacity to bind anti-T1R-like ligand II antibody. Such cells maybe cultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 μg/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable mycloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent mycloma cell line (SP₂O), available from the AmericanType Culture Collection (ATCC) (Rockville, Md., USA). After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution as described by Wands et al.,Gastroenterology 80:225–232 (1981); Harlow & Lane, infra, Chapter 7. Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the T1R-likeligand II antigen.

Alternatively, additional antibodies capable of binding to the T1R-likeligand II antigen may be produced in a two-step procedure through theuse of anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and therefore it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, T1R-like ligand II specific antibodies are used to immunizean animal, preferably a mouse. The splenocytes of such an animal arethen used to produce hybridoma cells, and the hybridoma cells arescreened to identify clones which produce an antibody whose ability tobind to the T1R-like ligand II-specific antibody can be blocked by theT1R-like ligand II antigen. Such antibodies comprise anti-idiotypicantibodies to the T1R-like ligand II-specific antibody and can be usedto immunize an animal to induce formation of further T1R-like ligandII-specific antibodies.

It will be appreciated that Fab and F(ab′)₂ and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). Alternatively, T1R-like ligandII-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

Where in vivo imaging is used to detect enhanced levels of T1R-likeligand II for diagnosis in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).

Further suitable labels for the T1R-like ligand II-specific antibodiesof the present invention are provided herein. Examples of suitableenzyme labels include malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerolphosphate dehydrogenase, triose phosphate isomerase, peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹ Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging (Perkins et al., Eur. J. Nucl. Med. 10:296–301 (1985);Carasquillo et al., J. Nucl. Med. 28:281–287 (1987)). For example, ¹¹¹Incoupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumorous tissues, particularly the liver,and therefore enhances specificity oftumor localization (Esteban et al.,J. Nucl. Med. 28:861–870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and Fe.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al. (Clin. Chim. Acta 70:1–31 (1976)), andSchurs et al. (Clin. Chim. Acta 81:1–40 (1977)). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of an T1R-like ligand II gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose. Typically, in accordance with routine procedures forchromosome mapping, some trial and error may be necessary to identify agenomic probe that gives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15–25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified portion.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of portions from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Pharmaceutical Compositions and Therapeutic Administration

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like.

The composition, if desired, can also contain minor amounts ofwetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the comnpound,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429–4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527–1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353–365 (1989); Lopez-Berestein, ibid., pp. 317–327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used todeliver the compositions of the invention (see Langer, supra; Sefton,CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). Inanother embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983);see also Levy et al., Science 228:190 (1985); During et al., Ann.Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115–138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527–1533 (1990)).

T1R-Like ligand II compositions of the invention are also suitablyadministered by sustained-release systems. Suitable examples ofsustained-release compositions include suitable polymeric materials(such as, for example, semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules), suitable hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, and sparingly soluble derivatives (such as, forexample, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547–556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167–277 (1981), and Langer, Chem. Tech. 12:98–105 (1982)),ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release compositions also include liposomally entrappedcompositions of the invention (see generally, Langer, Science249:1527–1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317–327 and 353–365 (1989)). Liposomes containing T1R-Likeligand II polypeptide may be prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688–3692(1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030–4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200–800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal T1R-Like ligand II polypeptide therapy.

In a specific embodiment where the compound of the invention is anucleic acid encoding a polypeptide, antibody, antagonist, agonist,protein, or fragment or variant thereof, the nucleic acid can beadministered in vivo to promote expression of its encoded protein, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., by use of aretroviral vector (see U.S. Pat. No. 4,980,286), by direct injection, byuse of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont),or coating with lipids or cell-surface receptors or transfecting agents,or by administering it in linkage to a homeobox-like peptide which isknown to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad.Sci. USA 88:1864–1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

T1R-like ligand II polynucleotide, polypeptide, antibody, antagonist,agonist, or fragment or variant thereof of the invention may beadministered using any method known in the art, including, but notlimited to, direct needle injection at the delivery site, intravenousinjection, topical administration, catheter infusion, biolisticinjectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. T1R-like ligand II molecules of the invention may beadministered as part of a pharmaceutical composition, described in moredetail herein. Methods of delivering T1R-like ligand II molecules of theinvention are known in the art and described in more detail herein.

The pharmaceutical compositions of the present invention may beadministered, for example, by the parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, trans-dermal, or buccal routes.Alternatively, or concurrently, administration may be oral. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

Compositions within the scope of this invention include all compositionswherein a T1R-like ligand II polynucleotide, polypeptide, antibody,agonist, antagonist or variant or fragment thereof is contained in anamount effective to achieve its intended purpose. While individual needsvary, determination of optimal ranges of effective amounts of eachcomponent is within the skill of the art. The effective dose is afunction of the individual T1R-like ligand II polynucleotide,polypeptide, antibody, agonist, antagonist or fragment or variantthereof, the presence and nature of a conjugated therapeutic agent (seeherein), the patient and his clinical status, and can vary from about 10ng/kg body weight to about 100 mg/kg body weight. The preferred dosagescomprise 0.1 to 10 mg/kg body wt.

Preparations of a T1R-like ligand II polynucleotide, polypeptide,antibody, agonist, antagonist or fragment or variant thereof, forparenteral administration, such as in detectably labeled form forimaging or in a free or conjugated form for therapy, include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propyleneglycol, polyethyleneglycol,vegetable oil such as olive oil, and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia, parenteral vehicles including sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present, such as, for example,antimicrobials, anti-oxidants, chelating agents, and inert gases and thelike. See, generally, Remington's Pharmaceutical Science, 16th ed., MackPublishing Co., Easton, Pa., 1980.

As a general proposition, the total pharmaceutically effective amount ofa T1R-like ligand II administered parenterally per dose will be in therange of about 0.01 ng/kg/day to 10 μg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 1.0 ng/kg/day, andmost preferably for humans between about 1.0 to 100 ng/kg/day. If givencontinuously, the T1R-like ligand II is typically administered at a doserate of about 0.01 ng/kg/hour to about 100 ng/kg/hour, either by 1–4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

A course of T1R-like ligand II polypeptide treatment to affect theimmune system appears to be optimal if continued longer than a certainminimum number of days, 7 days in the case of the mice. The length oftreatment needed to observe changes and the interval following treatmentfor responses to occur appears to vary depending on the desired effect.

For parenteral administration, in one embodiment, the T1R-like ligand IIpolypeptide is formulated generally by mixing it at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrastemal,subcutaneous and intraarticular injection and infusion.

Generally, the formulations are prepared by contacting the T1R-likeligand II polypeptide uniformly and intimately with liquid carriers orfinely divided solid carriers or both. Then, if necessary, the productis shaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The T1R-like ligand II is typically formulated in such vehicles at aconcentration of about 0.001 ng/ml to 500 ng/ml, preferably 0.1–10ng/ml, at a pH of about 3 to 8. It will be understood that the use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of T1R-like ligand II salts.

T1R-like ligand II tobe used fortherapeutic administration must besterile. Sterilityis readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic T1R-likeligand II compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

T1R-like ligand II ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous T1R-like ligand II solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized T1R-like ligand II usingbacteriostatic Water-for-Injection.

For example, satisfactory results are obtained by oral administration ofa polypeptide having T1R-like ligand II activity in dosages on the orderof from 0.05 to 5000 ng/kg/day, preferably 0.1 to 1000 ng/kg/day, morepreferably 10 to 100 ng/kg/day, administered once or, in divided doses,1 to 4 times per day. On administration parenterally, for example byi.v. drip or infusion, dosages on the order of from 0.01 to 500ng/kg/day, preferably 0.05 to 100 ng/kg/day and more preferably 0.1 to50 ng/kg/day can be used. Suitable daily dosages for patients are thuson the order of from 2.5 ng to 250 μg p.o., preferably 5 ng to 50 μgp.o., more preferably 50 ng to 12.5 μg p.o., or on the order of from 0.5ng to 25 μg i.v., preferably 2.5 ng to 500 μg i.v. and more preferably 5ng to 2.5 μg i.v.

The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the IL-1, IL-1R or T1R-likeligand II family, chemotherapeutic agents, antivirals, antibiotics,steroidal and non-steroidal anti-inflammatories, conventionalimmunotherapeutic agents, cytokines, chemokines and/or growth facotrs.Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The invention also encompasses combining the polynucleotides and/orpolypeptides of the invention (and/or agonists or antagonists thereof)with other proposed or conventional hematopoietic therapies. Thus, forexample, the polynucleotides and/or polypeptides of the invention(and/or agonists or antagonists thereof) can be combined with compoundsthat singly exhibit erythropoietic stimulatory effects, such aserythropoietin, testosterone, progenitor cell stimulators, insulin-likegrowth factor, prostaglandins, serotonin, cyclic AMP, prolactin, andtriiodothyzonine. Also encompassed are combinations of the compositionsof the invention with compounds generally used to treat aplastic anemia,such as, for example, methenolene, stanozolol, and nandrolone; to treatiron-deficiency anemia, such as, for example, iron preparations; totreat malignant anemia, such as, for example, vitamin B₁₂ and/or folicacid; and to treat hemolytic anemia, such as, for example,adrenocortical steroids, e.g., corticoids. See e.g., Resegotti et al.,Panminerva Medica, 23:243–248 (1981); Kurtz, FEBS Letters, 14a:105–108(1982); McGonigle et al., Kidney Int., 25:437–444 (1984); andPavlovic-Kantera, Expt. Hematol., 8(supp. 8)283–291(1980), the contentsof each of which are hereby incorporated by reference in theirentireties.

Compounds that enhance the effects of or synergize with erythropoietinare also useful as adjuvants herein, and include but are not limited to,adrenergic agonists, thyroid hormones, androgens, hepatic erythropoieticfactors, erythrotropins, and erythrogenins, See for e.g., Dunn, “CurrentConcepts in Erythropoiesis”, John Wiley and Sons (Chichester, England,1983); Kalmani, Kidney Int., 22:383–391 (1982); Shahidi, New Eng. J.Med., 289:72–80 (1973); Urabe et al., J. Exp. Med., 149:1314–1325(1979); Billat et al., Expt. Hematol., 10:133–140 (1982); Naughton etal., Acta Haemat, 69:171–179 (1983); Cognote et al. in abstract 364,Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec, Jul.1–7, 1984); and Rothman et al., 1982, J. Surg. Oncol., 20:105–108(1982). Methods for stimulating hematopoiesis comprise administering ahematopoietically effective amount (i.e., an amount which effects theformation of blood cells) of a pharmaceutical composition containingpolynucleotides and/or poylpeptides of the invention (and/or agonists orantagonists thereof) to a patient.

The polynucleotides and/or polypeptides of the invention and/or agonistsor antagonists thereof is administered to the patient by any suitabletechnique, including but not limited to, parenteral, sublingual,topical, intrapulmonary and intranasal, and those techniques furtherdiscussed herein. The pharmaceutical composition optionally contains oneor more members of the group consisting of erythropoietin, testosterone,progenitor cell stimulators, insulin-like growth factor, prostaglandins,serotonin, cyclic AMP, prolactin, triiodothyzonine, methenolene,stanozolol, and nandrolone, iron preparations, vitamin B₁₂, folic acidand/or adrenocortical steroids.

In additional prefered embodiments, the compositions of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with thecompositions of the invention included, but are not limited to LEUKINE(SARGRAMOSTIM™) and NEUPOGEN (FILGRASTIM™).

In one embodiment, the compositions of the invention are administered incombination with other members of the IL1- and IL1R-like family.Molecules that may be administered with the compositions of theinvention include, but are not limited to, IL-1α, IL-1β, IL-1R andIL1-Ra.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

In one embodiment, the compositions of the invention are administered incombination with one or more chemokines. In specific embodiments, thecompositions of the invention are administered in combination with anα(C×C) chemokine or nucleic acid encoding an α chemokine selected fromthe group consisting of γ interferon inducible protein-10 (γIP-10),interleukin-8 (IL-8), platelet factor-4 (PF4), neutrophil activatingprotein (NAP-2), GRO-α, GRO-β, GRO-γ, neutrophil-activating peptide(ENA-78), granulocyte chemoattractant protein-2 (GCP-2), and stromalcell-derived factor-1 (SDF-1, or pre-B cell stimulatory factor (PBSF));and/or a β(CC) chemokine or nucleic acid encoding a β chemokine selectedfrom the group consisting of: RANTES (regulated on activation, normal Texpressed and secreted), macrophage inflammatory protein-1α (MIP-1α),macrophage inflammatory protein-1β (MIP-1β), monocyte chemotacticprotein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2), monocytechemotactic protein-3 (MCP-3), monocyte chemotactic protein-4 (MCP-4)macrophage inflammatory protein-1γ (MIP-1γ), macrophage inflammatoryprotein-3α (MIP-3α), macrophage inflammatory protein-3β (MIP-3β),macrophage inflammatory protein-4 (MIP-4/DC-CK-1/PARC), eotaxin, Exodus,and I-309; and/or the γ(C) chemokine, or nucleic acid encoding the γchemokine, lymphotactin.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259–268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

Additionally, the compositions of the invention may be administeredalone or in combination with other therapeutic regimens, including butnot limited to, radiation therapy. Such combinatorial therapy may beadministered sequentially and/or concomitantly.

Diagnostic Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976–985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087–3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subj ect and theimaging system used wll determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Treatment of T1R-Like Ligand II Disorders

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention. In a preferred aspect, thecompound is substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Thesubject is preferably an animal, including but not limited to animalssuch as cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human.

It is believed by the present inventors that T1R-like ligand IIpolypeptides of the present invention share biological activities withinterleukin-1 (IL-1) and the T1R ligand. Thus, the T1R-like ligand IIpolypeptide, antibody, antagonist, agonist, protein, or fragment orvariant thereof, can be exogenously added to cells, tissues, or the bodyof an individual to produce a therapeutic effect. In particular,disorders caused by a decrease in the standard level of T1R-like ligandII protein activity can be treated by administering an effective amountof a T1R-like ligand II polypeptide or agonist of the invention.Preferably, a pharmaceutical composition is administered comprising anamount of an isolated T1R-like ligand II polypeptide or agonist of theinvention effective to increase the T1R-like ligand II protein activity.Disorders where such a therapy would likely be effective are discussedabove and herein.

As shown below in Example 18, T1R-like ligand II stimulatesproliferation of CD34+ cells. Thus, it is expected that T1R-like ligandII will stimulate other hematopoietic stem cells and cells originatingfrom hematopoietic stem cells.

A hematopoietic stem cell is a developmentally multipotent stem cellfound in hematopoietic, or blood-forming tissue. It has the potential tomature into a mature blood cell through synergism betweenlineage-specific and multilineage growth factors. Tissues containinghematopoietic cells are found in various body locations including forexample, bone marrow, spleen, and thymus. In the process ofhematopoiesis, distinct populations of progenitor cells arise from moreprimitive, undifferentiated stem cells. Subsequent developmentaleventually results in differentiation of mature classes of blood cells(for example, granulocytes, monocytes, eosinophils, megakaryocytes, andmast cells) from progenitor cells.

One of ordinary skill will appreciate that effective amounts of aT1R-like ligand II polynucleotide, polypeptide, antibody, antagonist,agonist, or fragment or variant thereof can be determined empiricallyfor each condition where administration of a such is indicated. Thepolypeptide having T1R-like ligand II activity or antibody, agonist,antagonist, or fragment or variant thereof modulating such activity, canbe administered in pharmaceutical compositions in combination with oneor more pharmaceutically acceptable carriers, diluents and/orexcipients. It will be understood that, when administered to a humanpatient, the total daily usage of the pharmaceutical compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular patient will depend upon a variety offactors including the type and degree of the response to be achieved;the specific composition an other agent, if any, employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thecomposition; the duration of the treatment; drugs (such as achemotherapeutic agent) used in combination or coincidental with thespecific composition; and like factors well known in the medical arts.

The T1R-like ligand II composition to be used in the therapy will alsobe formulated and dosed in a fashion consistent with good medicalpractice, taking into account the clinical condition of the individualpatient (especially the side effects of treatment with T1R-like ligandII alone), the site of delivery of the T1R-like ligand II composition,the method of administration, the scheduling of administration, andother factors known to practitioners. An “effective amount” of aT1R-like ligand II polypeptide for purposes herein is thus determined bysuch considerations. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like.

T1R-Like ligand II polynucleotides and polypeptides of the invention maybe used in developing treatments for any disorder mediated (directly orindirectly) by defective, or insufficient amounts of T1R-Like ligand II.T1R-Like ligand II polypeptides may be administered to a patient (e.g.,mammal, preferably human) afflicted with such a disorder. Alternatively,a gene therapy approach may be applied to treat such disorders.Disclosure herein of T1R-Like ligand II nucleotide sequences permits thedetection of defective T1R-Like ligand II genes, and the replacementthereof with normal T1R-Like ligand II encoding genes. Defective genesmay be detected in in vitro diagnostic assays, and by comparison of theT1R-Like ligand II nucleotide sequence disclosed herein with that of aT1R-Like ligand II gene derived from a patient suspected of harboring adefect in this gene.

In another embodiment, the polypeptides of the present invention areused as a research tool for studying the biological effects that resultfrom inhibiting T1R-Like ligand II ligand interactions on different celltypes. T1R-Like ligand II polypeptides also may be employed in in vitroassays for detecting T1R-Like ligand II or T1R-Like ligand II ligand orthe interactions thereof.

T1R-Like ligand II polynucleotides or polypeptides, or agonists orantagonists of T1R-Like ligand II, may be useful in treatingdeficiencies or disorders of the immune system, by activating orinhibiting the proliferation, differentiation, or mobilization(chemotaxis) of immune cells. Immune cells develop through a processcalled hematopoiesis, producing myeloid (platelets, red blood cells,neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cellsfrom pluripotent stem cells. The etiology of these immune deficienciesor disorders may be genetic, somatic, such as cancer or some autoimmunedisorders, acquired (e.g., by chemotherapy or toxins), or infectious.Moreover, T1R-Like ligand II polynucleotides or polypeptides, oragonists or antagonists of T1R-Like ligand II, can be used as a markeror detector of a particular immune system disease or disorder. It isbelieved that T1R-like ligand II stimulates proliferation and/ordifferentiation of cells of hematopoietic origin, eg. myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells. As shown below, T1R-like ligand IIpolypeptides can be used to stimulate the proliferation of CD34+ cells.

By the invention, disorders caused by enhanced levels of T1R-like ligandII protein activity can be treated by administering an effective amountof an antagonist of a T1R-like ligand II polypeptide of the invention.Therefore, antibodies (preferably monoclonal) or antibody fragments thatbind a T1R-like ligand II polypeptide of the present invention areuseful in treating T1R-like ligand II-related disorders as are solubleT1R-like ligand II proteins, such as the extracellular domain, whichcompetes with the intact protein for binding to the T1R-like ligand IIreceptor. Such antibodies and/or soluble T1R-like ligand II proteins arepreferably provided in pharmaceutically acceptable compositions.

The antibodies described herein may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hemopoietic growth factors, etc., which serve to increasethe number or activity of effector cells which interact with theantibodies.

T1R-Like ligand II polynucleotides or polypeptides, or agonists orantagonists of T1R-Like ligand II, may be useful in treating ordetecting deficiencies or disorders of hematopoietic cells. T1R-LikeLigand II polynucleotides or polypeptides, or agonists or antagonists ofT1R-Like Ligand II, could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat those disorders associated with a decreasein certain (or many) types hematopoietic cells. Examples of immunologicdeficiency syndromes include, but are not limited to: blood proteindisorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxiatelangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIVinfection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome,lymphopenia, phagocyte bactericidal dysfunction, severe combinedimmunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,thrombocytopenia, or hemoglobinuria.

In a specific embodiment, polynucleotides and/or polypeptides of theinvention and/or angonists and/or antagonists thereof may be used toincrease the concentration of blood cells in individuals in need of suchincrease (i.e., in hematopoietin therapy). Conditions that may beameliorated by administering the compositions of the invention include,but are not limited to, neutropenia, anemia, and thrombocytopenia.

In a specific embodiment, the polynucleotides and/or polypeptides of theinvention (and/or agonists or antagonists thereof) are used inerythropoietin therapy, which is directed toward supplementing theoxygen carrying capacity of blood. Polynucleotides and/or polypeptidesof the invention (and/or agonists or antagonists thereof) may be used totreat or prevent diseases or conditions in patients generally requiringblood transfusions, such as, for example, trauma victims, surgicalpatients, dialysis patients, and patients with a variety of bloodcomposition-affecting disorders, such as, for example, hemophilia,cystic fibrosis, pregnancy, menstrual disorders, early anemia ofprematurity, spinal cord injury, aging, various neoplastic diseasestates, and the like. Examples of patient conditions that requiresupplementation of the oxygen carrying capacity of blood and which arewithin the scope of this invention, include,but are not limited to:treatment of blood disorders characterized by low or defective red bloodcell production, anemia associated with chronic renal failure,stimulation of reticulocyte response, development of ferrokineticeffects (such as plasma iron turnover effects and marrow transit timeeffects), erythrocyte mass changes, stimulation of hemoglobin Csynthesis, and increasing levels of hematocrit invertebrates. Theinvention also provides for treatment to enhance the oxygen-carryingcapacity of an individual, such as for example, an individualencountering hypoxic environmental conditions.

As further described herein, the a polypeptide, polynucleotide, agonist,or antagonist of the present invention may be employed to stimulategrowth and differentiation of hematopoietic cells and bone marrow cellseither when used alone or when used in combination with other cytokines.

The polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof, can also be employed to inhibit theproliferation and differentiation of hematopoietic cells and thereforemay be employed to protect bone marrow stem cells from chemotherapeuticagents during chemotherapy. This antiproliferative effect may allowadministration of higher doses of chemotherapeutic agents and,therefore, more effective chemotherapeutic treatment.

The polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof, may also be employed for the expansion ofimmature hematopoeitic progenitor cells, for example, granulocytes,macrophages or monocytes, by temporarily preventing theirdifferentiation. These bone marrow cells may be cultured in vitro. Thus,T1R-Like ligand II polypeptides, polynucleotides , or agonists orantagonists thereof, may be useful as a modulator of hematopoietic stemcells in vitro for the purpose of bone marrow transplantation and/orgene therapy. Since stem cells are rare and are most useful forintroducing genes into for gene therapy, T1R-Like ligand II can be usedto isolate enriched populations of stem cells. Stem cells can beenriched by culturing cells in the presence of cytotoxins, such as 5-Fu,which kills rapidly dividing cells, where as the stem cells will beprotected by T1R-Like Ligand II. These stem cells can be returned to abone marrow transplant patient or can then be used for transfection ofthe desired gene for gene therapy. In addition, T1R-like ligand II canbe injected into animals which results in the release of stem cells fromthe bone marrow of the animal into the peripheral blood. These stemcells can be isolated for the purpose of autologous bone marrowtransplantation or manipulation for gene therapy. After the patient hasfinished chemotherapy or radiation treatment, the isolated stem cellscan be returned to the patient.

T1R-like ligand II also may have a role in vesicle trafficking, and thusmay be associated with disorders of abnormal vesicle trafficking,including endocrine, secretory, inflammatory, and gastrointestinaldisorders, and in the development of cancers, particularly thoseinvolving secretory and gastrointestinal tissues.

Therefore, in one embodiment, T1R-like ligand II polynucleotides,polypeptides, antibodies, agonists, antagonists and/or fragments orvariants thereof may be administered to a subject to treat disordersassociated with abnormal vesicle trafficking. Such disorders mayinclude, but are not limited to, glucose-galactose malabsorptionsyndrome, hypercholesterolemia, diabetes insipidus, hyper- andhypoglycemia, goiter, Cushing's disease;- gastrointestinal disordersincluding ulcerative colitis, gastric and duodenal ulcers; and otherconditions associated with abnormal vesicle trafficking includingallergies including hay fever; osteoarthritis; and Chediak-Higashisyndrome.

Cancer cells secrete excessive amounts of hormones or other biologicallyactive peptides. Therefore, in another embodiment, polynucleotides,polypeptides, antibodies, agonists, antagonists and/or fragments orvariants thereof of T1R-like ligand II may be administered to a subjectto treat or prevent cancer, including, but not limited to, cancers ofglands, tissues, and organs involved in secretion or absorption,including prostate, pancreas, lung, tongue, brain, breast, bladder,adrenal gland, thyroid, liver, uterus, kidney, testes, and organs of thegastrointestinal tract including small intestine, colon, rectum, andstomach. In particular, antibodies which are specific for T1R-likeligand II may be used directly as an antagonist, or indirectly as atargeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissue which express T1R-like ligand II. Additional preferredembodiments of the invention include, but are not limited to, the use ofT1R-like ligand II polynucleotides, polypeptides, and functionalagonists thereof, in the following applications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody production(e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.

Administration to an animal (including, but not limited to, those listedabove, and also including transgenic animals) incapable of producingfunctional endogenous antibody molecules or having an otherwisecompromised endogenous immune system, but which is capable of producinghuman immunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO 98/24893, WO/9634096, WO/9633735, andWO/9110741.

A vaccine adjuvant that enhances immune responsiveness to specificantigen. In a specific embodiment, the vaccine adjuvant is a polypeptidedescribed herein. In another specific embodiment, the vaccine adjuvantis a polynucleotide described herein (i.e., the polynucleotide is agenetic vaccine adjuvant). As discussed herein, polynucleotides may beadministered using techniques known in the art, including but notlimited to, liposomal delivery, recombinant vector delivery, injectionof naked DNA, and gene gun delivery.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include virus and virus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g.,hepatitis B). In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response to avirus, disease, or symptom selected from the group consisting of:HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese Bencephalitis, Influenza A and B, Parainfluenza, Measles,Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpessimplex, and yellow fever. In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to the HIV gp120 antigen.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses.Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacteria or fungus, disease, or symptom selectedfrom the group consisting of: tetanus, Diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae,Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasiticimmune responses that may be enhanced using the compositions of theinvention as an adjuvant, include parasite and parasite associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a parasite. In anotherspecific embodiment, the compositions of the invention are used as anadjuvant to enhance an immune response to Plasmodium (malaria).

As a stimulator of B cell responsiveness to pathogens.

As an agent that elevates the immune status of an individual prior totheir receipt of immunosuppressive therapies.

As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrations.

As an agent to accelerate recovery of immunocompromised individuals.

As an agent to boost immunoresponsiveness among aged populations.

As an immune system enhancer prior to, during, or after bone marrowtransplant and/or other transplants (e.g., allogeneic or xenogeneicorgan transplantation). With respect to transplantation, compositions ofthe invention may be administered prior to, concomitant with, and/orafter transplantation. In a specific embodiment, compositions of theinvention are administered after transplantation, prior to the beginningof recovery of T-cell populations. In another specific embodiment,compositions of the invention are first administered aftertransplantation after the beginning of recovery of T cell populations,but prior to full recovery of B cell populations.

As an agent to boost immunoresponsiveness among immunodeficientindividuals. B cell immunodeficiencies that may be ameliorated ortreated by administering the polypeptides or polynucleotides of theinvention, or agonists thereof, include, but are not limited to, severecombined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosinedeaminase deficiency (ADA deficiency), X-linked agammaglobulinemia(XLA), Bruton's disease, congenital agammaglobulinemia, X-linkedinfantile agammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVI) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymophoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

In a specific embodiment, polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat or ameliorateselective IgA deficiency. In another specific embodiment, polypeptidesor polynucleotides of the invention, or agonists thereof, isadministered to treat or ameliorate ataxia-telangiectasia. In anotherspecific embodiment, polypptides or polynucleotides of the invention, oragonists thereof, is administered to treat or ameliorate common-variable immunodeficiency. In another specific embodiment, polypeptidesor polynucleotides of the invention, or agonists thereof, isadministered to treat or ameliorate X-linked agammaglobulinemia. Inanother specific embodiment, polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat or amelioratesevere combined immunodeficiency (SCID). In another specific embodiment,polypeptides or polynucleotides of the invention, or agonists thereof,is administered to treat or ameliorate Wiskott-Aldrich syndrome. Inanother specific embodiment, polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat or amelioratesevere combined immunodeficiency (SCID). In another specific embodiment,polypeptides or polynucleotides of the invention, or agonists thereof,is administered to treat or ameliorate X-linked Ig deficiency with hyperIgM. T cell immunodeficiencies that may be ameliorated or treated byadministering the polypeptides or polynucleotides of the invention, oragonists thereof, include, but are not limited to, DiGeorge anomaly(thymic hypoplasia), chronic mucocutaneous candidiasis, natural killercell deficiency, idiopathic CD4+ T-lymphocytopenia, immunodeficiencywith predominant T-cell defect, and unspecified ummunodeficiency of cellmediated immunity.

Phagocyte disorder related immunodeficiencies that may be ameliorated ortreated by administering the polypeptides or polynucleotides of theinvention, or agonists thereof, include, but are not limited to,Hyperimmunoglobulinemia E syndrome (HIE), leukocyte adhesion defect type1, chronic granulomatous disease, neutrophil G6PD deficiency,Chediak-Higashi syndrome, splenic deficiency syndromes, andmyeloperoxidase deficiency. Complement disorder relatedimmunodeficiencies that may be ameliorated or treated by administeringthe polypeptides or polynucleotides o the invention, or agoniststhereof, include, but are not limited to, 1q deficiency, C1–C9deficiencies, and C2 deficiencies

As an agent to boost immunoresponsiveness among individuals having anacquired loss of B cell and/or T cell function. Conditions resulting inan acquired loss of B cell function that may be ameliorated or treatedby administering the polypeptides or polynucleotides of the invention,or agonists thereof, include, but are not limited to, HIV Infection,AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia(CLL).

As an agent to boost immunoresponsiveness among individuals having atemporary immune deficiency. Conditions resulting in a temporary immunedeficiency that may be ameliorated or treated by administering thepolypeptides or polynucleotides of the invention, or agonists thereof,include, but are not limited to, recovery from viral infections (e.g.,influenza), conditions associated with malnutrition, recovery frominfectious mononucleosis, or conditions associated with stress, recoveryfrom measles, recovery from blood transfusion, recovery from surgery.

As a regulator of antigen presentation by monocytes, dendritic cells, Tcells and/or B-cells. In one embodiment, polypeptides (in soluble,membrane-bound or transmembrane forms) or polynucleotides enhanceantigen presentation or antagonize antigen presentation in vitro or invivo. Moreover, in related embodiments, said enhancement orantagonization of antigen presentation may be useful as an anti-tumortreatment or to modulate the immune system.

As an agent to direct an individuals immune system towards developmentof a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

As B cell, monocytic cell, and/or T cell specific binding protein towhich specific activators or inhibitors of cell growth may be attached.The result would be to focus the activity of such activators orinhibitors onto normal, diseased, or neoplastic cell populations.

As a stimulator of B cell production in pathologies such as AIDS,chronic lymphocyte disorder and/or Common Variable Immunodificiency;

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceT1R-like ligand II mediated responses.

As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell and/or T cell representation and thusaccelerate recovery.

As a means of regulating secreted cytokines that are elicited byT1R-like ligand II.

As a means to modulate IgE concentrations in vitro or in vivo.Additionally, T1R-like ligand II polypeptides or polynucleotides of theinvention, or agonists thereof, may be used to treat or preventIgE-mediated allergic reactions. Such allergic reactions include, butare not limited to, asthma, rhinitis, and eczema.

All of the above described applications as they may apply to veterinarymedicine.

Antagonists of T1R-like ligand II include binding and/or inhibitoryantibodies, antisense nucleic acids, ribozymes or soluble forms of theT1R-like ligand II receptor(s). These would be expected to reverse manyof the activities of the ligand described above as well as find clinicalor practical application as:

A means of blocking various aspects of immune responses to foreignagents or self. Examples include autoimmune disorders such as lupus, andarthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens.

A therapy for preventing the B cell proliferation and/or Ig secretionassociated with autoimmune diseases such as idiopathic thrombocytopenicpurpura, systemic lupus erythramatosus and MS.

An inhibitor of graft versus host disease or transplant rejection.

A therapy for B cell T cell and/or monocyitic malignancies, such as, forexample, ALL, Hodgkins disease, non-Hodgkins lymphoma, Chroniclymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt'slymphoma, and EBV-transformed diseases.

A therapy for chronic hypergammaglobulinemeia evident in such diseasesas monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonalgammopathies, andplasmacytomas.

A therapy for decreasing cellular proliferation of Large B-cellLymphomas.

A means of decreasing the involvement of B cells and Ig associated withChronic Myelogenous Leukemia.

An immunosuppressive agent(s).

T1R-like ligand II polypeptides or polynucleotides of the invention, orantagonists may be used to modulate IgE concentrations in vitro or invivo. In another embodiment, administration of polypeptides orpolynucleotides of the invention, or antagonists thereof, may be used totreat or prevent IgE-mediated allergic reactions including, but notlimited to, asthma, rhinitis, and eczema.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit the chemotaxisand activation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T-cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in certain auto-immuneand chronic inflammatory and infective diseases. Examples of auto-immunediseases include multiple sclerosis, and insulin-dependent diabetes. Theantagonists may also be employed to treat infectious diseases includingsilicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing therecruitment and activation of mononuclear phagocytes. They may also beemployed to treat idiopathic hyper-eosinophilic syndrome by preventingeosinophil production and migration. Endotoxic shock may also be treatedby the antagonists by preventing the migration of macrophages and theirproduction of the polypeptides of the present invention. The antagonistsmay also be employed for treating atherosclerosis, by preventingmonocyte infiltration in the artery wall. The antagonists may also beemployed to treat histamine-mediated allergic reactions andimmunological disorders including late phase allergic reactions, chronicurticaria, and atopic dermatitis by inhibiting chemokine-induced mastcell and basophil degranulation and release of histamine.

IgE-mediated allergic reactions such as allergic asthma, rhinitis, andeczema may also be treated. The antagonists may also be employed totreat chronic and acute inflammation by preventing the attraction ofmonocytes to a wound area. They may also be employed to regulate normalpulmonary macrophage populations, since chronic and acute inflammatorypulmonary diseases are associated with sequestration of mononuclearphagocytes in the lung. Antagonists may also be employed to treatrheumatoid arthritis by preventing the attraction of monocytes intosynovial fluid in the joints of patients. Monocyte influx and activationplays a significant role in the pathogenesis of both degenerative andinflammatry arthropathies. The antagonists may be employed to interferewith the deleterious cascades attributed primarily to IL-1 and TNF,which prevents the biosynthesis of other inflammatory cytokines. In thisway, the antagonists may be employed to prevent inflammation. Theantagonists may also be employed to inhibit prostaglandin-independentfever induced by. The antagonists may also be employed to treat cases ofbone marrow failure, for example, aplastic anemia and myelodysplasticsyndrome. The antagonists may also be employed to treat asthma andallergy by preventing eosinophil accumulation in the lung. Theantagonists may also be employed to treat subepithelial basementmembrane fibrosis which is a prominent feature of the asthmatic lung.The antagonists may also be employed to treat lymphomas (e.g., one ormore of the extensive, but not limiting, list of lymphomas providedherein).

Moreover, T1R-Like Ligand II polynucleotides or polypeptides, oragonists or antagonists of T1R-Like Ligand II, can also be used tomodulate hemostatic (the stopping of bleeding) or thrombolytic activity(clot formation). For example, by increasing hemostatic or thrombolyticactivity, T 1R-Like Ligand II polynucleotides or polypeptides, oragonists or antagonists of T1R-Like Ligand II, could be used to treatblood coagulation disorders (e.g., afibrinogenemia, factordeficiencies), blood platelet disorders (e.g. thrombocytopenia), orwounds resulting from trauma, surgery, or other causes. Alternatively,T1R-Like Ligand II polynucleotides or polypeptides, or agonists orantagonists of T1R-Like Ligand II, that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.These molecules could be important in the treatment of heart attacks(infarction), strokes, or scarring.

T1R-Like Ligand II polynucleotides or polypeptides, or agonists orantagonists of T1R-Like Ligand II, may also be useful in treating ordetecting autoimmune disorders. Many auto immune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration ofT1R-Like Ligand II polynucleotides or polypeptides, or agonists orantagonists of T1R-Like Ligand II, that can inhibit an immune response,particularly the proliferation, differentiation, or chemotaxis ofT-cells, may be an effective therapy in treatiing or preventingautoimmune disorders or conditions associated with these disorders.

Examples of autoimmune disorders that can be treated, prevented ordetected using compositions of the invention include, but are notlimited, autoimmune diseases such as, for example, autoimmune hemolyticanemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia,hemolytic anemia, antiphospholipid syndrome, dermatitis, allergicencephalomyelitis, glomerulonephritis, Multiple Sclerosis, Neuritis,Ophthalmia, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-ManSyndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome,insulin dependent diabetes mellitis, and autoimmune inflammatory eyedisease.

Additional autoimmune disorders that can be treated, prevented ordetected using compositions of the invention include, but are notlimited to, autoimmune thyroiditis (i.e., Hashimoto's thyroiditis)(often characterized, e.g., by cell-mediated and humoral thyroidcytotoxicity), systemic lupus erhthematosus (often characterized, e.g.,by circulating and locally generated immune complexes), Goodpasture'ssyndrome (often characterized, e.g., by anti-basement membraneantibodies), Pemphigus (often characterized, e.g., by epidermalacantholytic antibodies), Receptor autoimmunities such as, for example,(a) Graves' Disease (often characterized, e.g., by TSH receptorantibodies), (b) Myasthenia Gravis (often characterized, e.g., byacetylcholine receptor antibodies), and (c) insulin resistance (oftencharacterized, e.g., by insulin receptor antibodies), autoimmunehemolytic anemia (often characterized, e.g., by phagocytosis ofantibody-sensitized RBCs), autoimmune thrombocytopenic purpura (oftencharacterized, e.g., by phagocytosis of antibody-sensitized platelets.

Additional autoimmune disorders that can be treated, prevented ordetected using compositions of the invention include, but are notlimited to, rheumatoid arthritis (often characterized, e.g., by immunecomplexes in joints), scleroderma with anti-collagen antibodies (oftencharacterized, e.g., by nucleolar and other nuclear antibodies), mixedconnective tissue disease (often characterized, e.g., by antibodies toextractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis(often characterized, e.g., by nonhistone ANA), pernicious anemia (oftencharacterized, e.g., by antiparietal cell, microsomes, and intrinsicfactor antibodies), idiopathic Addison's disease (often characterized,e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility(often characterized, e.g., by antispermatozoal antibodies),glomerulonephritis (often characterized, e.g., by glomerular basementmembrane antibodies or immune complexes), bullous pemphigoid (oftencharacterized, e.g., by IgG and complement in basement membrane),Sjogren's syndrome (often characterized, e.g., by multiple tissueantibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus(often characterized, e.g., by cell-mediated and humoral islet cellantibodies), and adrenergic drug resistance (including adrenergic drugresistance with asthma or cystic fibrosis) (often characterized, e.g.,by beta-adrenergic receptor antibodies).

Additional autoimmune disorders that can be treated, prevented ordetected using compositions of the invention include, but are notlimited to, chronic active hepatitis (often characterized, e.g., bysmooth muscle antibodies), primary biliary cirrhosis (oftencharacterized, e.g., by mitchondrial antibodies), other endocrine glandfailure (often characterized, e.g., by specific tissue antibodies insome cases), vitiligo (often characterized, e.g., by melanocyteantibodies), vasculitis (often characterized, e.g., by Ig and complementin vessel walls and/or low serum complement), post-MI (oftencharacterized, e.g., by myocardial antibodies), cardiotomy syndrome(often characterized, e.g., by myocardial antibodies), urticaria (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), atopicdermatitis (often characterized, e.g., by IgG and IgM antibodies toIgE), asthma (often characterized, e.g., by IgG and IgM antibodies toIgE), and many other inflammatory, granulamatous, degenerative, andatrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using anti-T1R-Like Ligand II.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by T1R-Like Ligand II polynucleotides or polypeptides, oragonists or antagonists of T1R-Like Ligand II. Moreover, these moleculescan be used to treat anaphylaxis, hypersensitivity to an antigenicmolecule, or blood group incompatibility.

T1R-Like Ligand II polynucleotides or polypeptides, or agonists orantagonists of T1R-Like Ligand II, may also be used to treat and/orprevent organ rejection or graft-versus-host disease (GVHD). Organrejection occurs by host immune cell destruction of the transplantedtissue through an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of T1R-Like Ligand IIpolynucleotides orpolypeptides, or agonists or antagonists of T1R-LikeLigand II, that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing organ rejection or GVHD.

Similarly, T1R-Like Ligand II polynucleotides or polypeptides, oragonists or antagonists of T1R-Like Ligand II, may also be used tomodulate inflammation. For example, T1R-Like Ligand II polynucleotidesor polypeptides, or agonists or antagonists of T1R-Like Ligand II, mayinhibit the proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat inflammatoryconditions, both chronic and acute conditions, including chronicprostatitis, granulomatous prostatitis and malacoplakia, inflammationassociated with infection (e.g., septic shock, sepsis, or systemicinflammatory response syndrome (SIRS)), ischemia-reperfusion injury,endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine induced lung injury,inflammatory bowel disease, Crohn's disease, or resulting from overproduction of cytokines (e.g., TNF or IL-1.)

Diseases associated with increased cell proliferation, survival, or theinhibition of, apoptosis that could be treated or detected by T1R-LikeLigand II polynucleotides or polypeptides, as well as antagonists oragonists of T1R-Like Ligand II, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection.

Thus, in preferred embodiments T1R-Like Ligand II polynucleotides orpolypeptides of the invention, or agonists or antagonists thereof, areused to treat, prevent, and/or diagnose autoimmune diseases and/orinhibit the growth, progression, and/or metastasis of cancers,including, but not limited to, those cancers disclosed herein, such as,for example, lymphocytic leukemias (including, for example, MLL andchronic lymphocytic leukemia (CLL)) and follicular lymphomas. In anotherembodiment T1R-Like Ligand II polynucleotides or polypeptides of theinvention are used to activate, differentiate or proliferate cancerouscells or tissue (e.g., B cell lineage related cancers (e.g., CLL andMLL), lymphocytic leukemia, or lymphoma) and thereby render the cellsmore vulnerable to cancer therapy (e.g., chemotherapy or radiationtherapy).

Moreover, in preferred embodiments, T1R-Like Ligand II polynucleotides,polypeptides, and/or antagonists of the invention are used to inhibitgrowth, progression, and/or metasis of cancers, in particular thoselisted above, and in the paragraphs that follow.

Additional diseases or conditions associated with increased cellsurvival that may be treated or detected by T1R-Like Ligand IIpolynucleotides or polypeptides, or agonists or antagonists of T1R-LikeLigand II, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lympsdhocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myclocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), and solid tumors including, but not limited to,polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors including, but not limited to, sarcomas andcarcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased cell death and/or decreased cellnumbers that may be treated or detected by T1R-Like Ligand IIpolynucleotides or polypeptides, or agonists or antagonists of T1R-LikeLigand II, include, but are not limited to, AIDS; neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration);myelodysplastic syndromes (such as aplastic anemia), ischemic injury(such as that caused by myocardial infarction, stroke and reperfusioninjury), toxin-induced liver disease (such as that caused by alcohol),septic shock, cachexia and anorexia. Thus, in preferred embodimentsT1R-Like Ligand II polynucleotides or polypeptides of the invention areused to treat, prevent, and/or diagnose the diseases and disorderslisted above and/or medical conditions associated with such disorders.

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of pathological cellproliferative neoplasias which include, but are not limited to: acutemyelogenous leukemias including acute monocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronicmyelogenous leukemias including chronic myelomonocytic leukemia, chronicgranulocytic leukemia, etc. Preferred mammals include monkeys, apes,cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

T1R-Like Ligand II polynucleotides or polypeptides, or agonists orantagonists thereof, can be used in the treatment of infectious agents.For example, by increasing the immune response, particularly increasingthe proliferation and/or differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, polynucleotides or polypeptides, or agonists orantagonists of, may also directly inhibit the infectious agent, withoutnecessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated by polynucleotides or polypeptides, oragonists of T1R-like ligand II. Examples of viruses, include, but arenot limited to the following DNA and RNA viruses and viral families:Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV,HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza A, Influenza B, and parainfluenza), Papiloma virus,Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such asSmallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae(HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus).Viruses falling within these families can cause a variety of diseases orsymptoms, including, but not limited to: arthritis, bronchiollitis,respiratory syncytial virus, encephalitis, eye infections (e.g.,conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunisticinfections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox,hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the commoncold, Polio, leukemia, Rubella, sexually transmitted diseases, skindiseases (e.g., Kaposi's, warts), and viremia. polynucleotidesorpolypeptides, or agonists or antagonists of, can be used to treat ordetect any of these symptoms or diseases. In specific embodiments,T1R-like ligand II polynucleotides, polypeptides, or agonists are usedto treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B).In an additional specific embodiment T1R-like ligand II polynucleotides,polypeptides, or agonists are used to treat patients nonresponsive toone or more other commercially available hepatitis vaccines. In afurther specific embodiment T1R-like ligand II polynucleotides,polypeptides, or agonists are used to treat AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated by polynucleotides or polypeptides, or agonistsor antagonists of, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi, Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseaes, skin diseases (e.g.,cellulitis, dermatocycoses), toxemia, urinary tract infections, woundinfections. polynucleotides or polypeptides, or agonists or antagonistsof, can be used to treat or detect any of these symptoms or diseases. Inspecific embodiments, T1R-like ligand II polynucleotides, polypeptides,or agonists thereof are used to treat: tetanus, Diptheria, botulism,and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated by polynucleotides or polypeptides, or agonists of, include, butnot limited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of, can be used to treat or detect any of thesesymptoms or diseases. In specific embodiments, T1R-like ligand IIpolynucleotides, polypeptides, or agonists thereof are used to treatmalaria.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or anti-T1R-like ligand IIantibodies associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs) to targeted cells, such as, forexample, cells expressing T1R-Like Ligand II receptor, or cellsexpressing the cell surface bound form of T1R-Like Ligand II. T1R-LikeLigand II polypeptides or anti-T1R-Like Ligand II antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., polypeptides or anti-T1R-likeligand II antibodies) that are associated with heterologous polypeptidesor nucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., T1R-like ligand IIpolypeptides or anti-T1R-like ligand II antibodies) in association withtoxins or cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for thespecific destruction of cells of B cell lineage (e.g., B cell relatedleukemias or lymphomas) by administering anti-T1R-like ligand IIantibodies and/or soluble T1R-like ligand II in association with toxinsor cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for thespecific destruction of cells of T cell lineage (e.g., T cell relatedleukemias or lymphomas) by administering anti-T1R-like ligand IIantibodies and/or soluble T1R-like ligand II in association with toxinsor cytotoxic prodrugs.

In another specific embodiment, the invention provides a method for thespecific destruction of cells of monocytic lineage (e.g., monocyticleukemias or lymphomas) by administering anti-T1R-like ligand IIantibodies and/or soluble T1R-like ligand II in association with toxinsor cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

An additional condition, disease or symptom that can be treatedbypolynucleotides or polypeptides, or agonists of, is osteomyelitis.

Preferably, treatment using polynucleotides or polypeptides, or agonistsof T1R-like ligand II, could either be by administering an effectiveamount of polypeptide to the patient, or by removing cells from thepatient, supplying the cells with polynucleotide, and returning theengineered cells to the patient (ex vivo therapy). Moreover, as furtherdiscussed herein, the polypeptide or polynucleotide can be used as anadjuvant in a vaccine to raise an immune response against infectiousdisease.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed to maintain organs before transplantationor for supporting cell culture of primary tissues. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may alsobe employed for inducing tissue of mesodermal origin to differentiate inearly embryos.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

As an agent to direct an individuals immune system towards developmentof a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

Expected Pleiotropic Biologic Effects of T1R-Like Ligand II

The T1R-like ligand II polypeptides of the present invention areexpected to have pleiotropic biological effects including many of thoseshown in Table 3 below. Similar biological effects have been shown forIL-1, particularly those associated with pancreatic endocrine tissue(Mandrup-Poulsen, T., et al., Cytokine 5:185 (1993)), thyroid glands(Rasmussen, A. K., Autoimmunity 16:141 (1993)),hypothalamic-pituitary-adrenal axis (Fantuzzi, G., & Ghezzi, P.,Mediator Inflamm. 2:263 (1993); Rivier, C., Ann. NY Acad. Sci. 697:97(1993); Rivier, C., & Rivest, S., Ciba. Found. Symp. 172:204 (1993)),fever (Coceani, F., “Fever: Basic Mechanisms and Management”, New York,N.Y., Raven (1991) p. 59), bone metabolism (Tatakis, D. N., J.Peridontol 64:416 (1993)), destruction of cartilage in the pathogenesisof rheumatoid arthritis (Arend, W. P., & Dayer, J. M., Arthritis Rheum33:305 (1990); Krane, S. M., et al., Ann. NY Acad. Sci. 580:340 (1990)),uterine implantation (Lewis, M. P., et al., Placenta 15:13 (1994)), andloss of lean body mass (Roubenoff, R., et al., J. Clin. Invest. 93:2379(1994)).

TABLE 3 POSSIBLE BIOLOGIC EFFECTS OF T1R-LIKE LIGAND II Effects ofsystemically injected T1R-like ligand II Fever; increased slow wavesleep; social depression; anorexia Hypotension; myocardial suppression;tachycardia; lactic acidosis Increased circulating nitric oxide;hypoaminoacidemia Hyperinsulinemia; hyperglycemia; hypoglycemiaStimulation of hypothalamic-pituitary-adrenal axis Release ofhypothalamic monoamines and neuropeptides Neutrophilia; increased marrowcellularity; increased platelets Increased hepatic acute phase proteinsynthesis Hypoferremia; hypozincemia; increased sodium excretionHyperlipidemia; increased muscle protein breakdown Hypoalbuminemia;decreased drug metabolism Increased metastases Increased nonspecificresistance to infection (pretreatment) Learning defects in offspringafter maternal IL-1 treatment Effects of locally injected T1R-likeligand II Infiltration of neutrophils into rabbits knee joint Increasedproteoglycan breakdown in rabbit knee joint Induction of uveitisfollowing intravitreal injection Angiogenesis in anterior chamber of eyeCellular infiltrate and cytokine induction in cerebral ventriclesNeutrophil and albumin influx into lungs after intratrachealinstillation Changes in immunologic responses Increased antibodyproduction (adjuvant effect) Increased lymphokine synthesis (IL-2, -3,-4, -5, -6, -7, -10 and -12) Increased IL-2 (β) receptor Development oftype 2 human T-cell clones Inhibition of tolerance to protein antigensEnhancement of spleen cell mitogenic response to LPS Effects of T1R-likeligand II on cultured cells or tissues Increased expression of ELAM-1,VCAM-1, ICAM-1 Cytotoxicity (apoptosis) of insulin-producing islet βcells Inhibition of thyroglobulin synthesis in thyrocytes Cartilagebreakdown, release of calcium from bone Increased release of arachidonicacid, prostanoids, and eicosanoids Increased mucus production andchloride flux in intestinal cells Enhancement in chloride flux (GABAAreceptor) in brain synaptosomes Proliferation of fibroblasts, smoothmuscle cells, messangial cells Growth inhibition of hair folliclesIncreased corticosterone synthesis by adrenals Increased HlV-1expression

Assays used: pancreatic endocrine tissue (Mandrup-Poulsen, T., et al.,Cytokine 5:185 (1993)), thyroid gland (Rasmussen, A. K., Autoimmunity16:141 (1993)), hypothalamic-pituitary-adrenal axis (Fantuzzi, G., &Ghezzi, P., Mediator Inflamm. 2:263 (1993); Rivier, C., Ann. NY Acad.Sci. 697:97 (1993); Rivier, C., & Rivest, S., Ciba. Found. Symp. 172:204(1993)), fever (Coceani, F., “Fever: Basic Mechanisms and Management”,New York, N.Y., Raven (1991) p. 59), bone metabolism (Tatakis, D. N., J.Peridontol 64:416 (1993)), destruction of cartilage in the pathogenesisof rheumatoid arthritis (Arend, W. P., & Dayer, J. M., Arthritis Rheum33:305 (1990); Krane, S. M., et al., Ann. NY Acad. Sci. 580:340 (1990)),uterine implantation (Lewis, M. P., et al., Placenta 15:13 (1994)), andloss of lean body mass (Roubenoff, R., et al., J. Clin. Invest. 93:2379(1994).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of T1R-Like Ligand II inE. coli

The DNA sequence encoding the mature, extracellular soluble portion ofT1R-like ligand II in the deposited cDNA clone is amplified using PCRoligonucleotide primers specific to the amino terminal sequences of theT1R-like ligand II and to vector sequences 3′ to the gene. Additionalnucleotides containing restriction sites to facilitate cloning are addedto the 5′ and 3′ sequences respectively.

One of ordinary skill in the art will understand that the full-length,mature T1R-like ligand II protein (amino acid about 1 to about 203 inSEQ ID NO:2) can be expressed in E. coli using suitable 5′ and 3′oligonucleotide primers.

The cDNA sequence encoding the extracellular domain of the full lengthT1R-like ligand II in the deposited clone is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene. The 5′ primer contains the sequence 5′ CGC CCA TGG CCG GCT TCA CACCTT CC 3′ (SEQ ID NO:4) containing the underlined Nco I site and 17nucleotides (nucleotides 131–147) of the T1R-like ligand II proteincoding sequence in FIGS. 1A–B (SEQ ID NO:1) beginning immediately afterthe signal peptide.

The 3′ primer has the sequence 5′ CGC AAG CTT TCA TCT ATC AAA GTT GCTTTC 3′ (SEQ ID NO:5) containing a Hind III restriction site followed bya stop codon and 18 nucleotides reverse and complementary to nucleotides619–636 of the T1R-like ligand II protein coding sequence in FIGS. 1A–B(SEQ ID NO:1).

The restriction sites are convenient to restriction enzyme sites in thebacterial expression vector pQE60, which are used for bacterialexpression in M15/rep4 host cells in these examples. (Qiagen, Inc.,Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibioticresistance (“Amp^(r)”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites.

The amplified T1R-like ligand II DNA and the vector pQE60 both aredigested with Nco I and Hind III and the digested DNAs are then ligatedtogether. Insertion of the T1R-like ligand II DNA into the restrictedpQE60 vector placed the T1R-like ligand II coding region downstream ofand operably linked to the vector's IPTG-inducible promoter and in-framewith an initiating AUG appropriately positioned for translation ofT1R-like ligand II.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressingT1R-like ligand II, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA was confirmed byrestriction analysis.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2×PBS.

Analysis of the preparation by standard methods of polyacrylamide gelelectrophoresis reveals that the preparation contains about 95% monomerT1R-like ligand II having the expected molecular weight of approximately26 kDa.

Example 2 Cloning and Expression of T1R-Like Ligand II in a BaculovirusExpression System

The cDNA sequence encoding the full length T1R-like ligand II in thedeposited clone is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene.

The 5′ primer has the sequence 5′ CGC GGA TCC GCC ATC ATG GGC GAC AAGATC TGG 3′ (SEQ ID NO:6) containing the underlined BamHI restrictionenzyme site followed by 18 nucleotides (nucleotides 55 to 72) of thesequence of the T1R-like ligand II protein in FIGS. 1A–B (SEQ ID NO:1).Inserted into an expression vector, as described herein, the 5′ end ofthe amplified fragment encoding T1R-like ligand II provides an efficientsignal peptide. An efficient signal for initiation oftranslation ineukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196: 947–950(1987) is appropriately located in the vector portion of the construct.

The 3′ primer has the sequence 5′ CGC GGT ACC TCA CAA TGT TAC GTA CTCTAG 3′ (SEQ ID NO:7) containing the underlined Asp 718 restriction sitefollowed by a stop codon and 18 nucleotides reverse and complementary tonucleotides 754–771 of the T1R-like ligand II coding sequence set out inFIGS. 1A–B (SEQ ID NO:1).

The cDNA sequence encoding the extracellular domain of the full lengthT1R-like ligand II in the deposited clone is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene.

The 5′ primer has the sequence 5′ CGC GGA TCC GCC ATC ATG GGC GAC AAGATC TGG 3′ (SEQ ID NO:6) containing the underlined BamHI restrictionenzyme site followed by 18 nucleotides (nucleotides 55–72) of thesequence encoding the T1R-like ligand II protein set out in FIGS. 1A–B(SEQ ID NO:1). Inserted into an expression vector, as described herein,the 5′ end of the amplified fragment encoding T1R-like ligand IIprovides an efficient signal peptide. An efficient signal for initiationof translation in eukaryotic cells, as described by Kozak, M., J. MolBiol. 196: 947–950 (1987) is appropriately located in the vector portionof the construct.

The 3′ primer has the sequence 5′ CGC GGT ACC TCA TCT ATC AAA GTT GCTTTC 3′ (SEQ ID NO:8) containing the underlined Asp 718 restriction sitefollowed by a stop codon and 18 nucleotides complementary and reverse tonucleotides 619–636 of the T1R-like ligand II coding sequence set out inFIGS. 1A–B (SEQ ID NO:1).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamH I and Asp 718 and againis purified on a 1% agarose gel. This fragment is designated herein F2.

The vector pA2 is used to express the T1R-like ligand II full length andextracellular domains of an T1R-like ligand II in the baculovirusexpression system, using standard methods, as described in Summers etal., A Manual of Methods for Baculovirus Vectors and Insect Cell CultureProcedures, Texas Agricultural Experimental Station Bulletin No. 1555(1987). The pA2 vector does not contain a signal peptide coding region.Thus, the T1R-like ligand II signal peptide is relied upon (nucleotides55–132 in SEQ ID NO:1; amino acids −26 to −1 SEQ ID NO:2).

If the T1R-like ligand II signal peptide does not result in efficientexpression of the T1R-like ligand II protein, the pA2-GP vector may beused instead of the pA2 vector. The signal peptide of AcMNPV gp67,including the N-terminal methionine, is located just upstream of a BamHIsite. One of ordinary skill in the art will understand that if thepA2-GP expression vector is used, the 5′ oligonucleotide used should notcontain sequence coding for the T1R-like ligand II signal peptide.Instead, the 5′ oligonucleotide should begin at nucleotide 131.

Both the pA2 and pA2-GP expression vectors contain the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV) followed by convenient restriction sites. The polyadenylationsite of the simian virus 40 (“SV40”) is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E. coli is inserted in the same orientationas the polyhedrin promoter and is followed by the polyadenylation signalof the polyhedrin gene. The polyhedrin sequences are flanked at bothsides by viral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate viable virus that express the clonedpolynucleotide.

Many other baculovirus vectors could be used in place of pA2 or pA2-GP,such as pAc373, pVL941 and pAcIMi provided, as those of skill readilywill appreciate, that construction provides appropriately locatedsignals for transcription, translation, trafficking and the like, suchas an in-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170:31–39, among others.

The plasmid is digested with the restriction enzyme BamnHI and Asp 718and then is dephosphorylated using calf intestinal phosphatase, usingroutine procedures known in the art. The DNA is then isolated from a 1%agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein “V2”.

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli HB101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human T1R-like ligand II gene by digesting DNA fromindividual colonies using BamHI and Asp 718 and then analyzing thedigestion product by gel electrophoresis. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBacT1R-like ligand II.

5 μg of the plasmid pBacT1R-like ligand II is co-transfected with 1.0 μgof a commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBacT1R-like ligand II are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9–10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Clonescontaining properly inserted T1R-like ligand II are identified by DNAanalysis including restriction mapping and sequencing. This isdesignated herein as V-T1R-like ligand II.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-T1R-like ligand II at a multiplicity of infection (“MOI”)of about 2 (about 1 to about 3). Six hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Gaithersburg). 42 hours later, 5μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then theyare harvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3 Cloning and Expression in Mammalian Cells

Most of the vectors used for the transient expression of the T1R-likeligand II protein gene sequence in mammalian cells should carry the SV40origin of replication. This allows the replication of the vector to highcopy numbers in cells (e.g. COS cells) which express the T antigenrequired for the initiation of viral DNA synthesis. Any other mammaliancell line can also be utilized for this purpose.

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV,HTLV-I, HIV-I and the early promoter of the cytomegalovirus (CMV).However, cellular signals can also be used (e.g. human actin promoter).Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Phannacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC67109). Mammalian host cells that could be used include, human Hela,283, H9 and Jurkat cells, mouse NIH3T3 and C 127 cells, Cos 1, Cos 7 andCV1, African green monkey cells, quail QC1-3 cells, mouse L cells andChinese hamster ovary cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) is a usefulmarker to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277–279 (1991); Bebbington et al., Bio/Technology 10:169–175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) cells are often used for the production ofproteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438–4470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521–530 (1985)). Multiple cloning sites, e.g. with therestriction enzyme cleavage sites BamHl, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

An expression plasmid is made by cloning a cDNA encoding T1R-like ligandII into the expression vector pcDNAI/Amp (which can be obtained fromInvitrogen, Inc.).

The expression vector pcDNAI/amp contains: (Dower, Colotta, F., et al.,Immunol Today 15:562 (1994)) an E. coli origin of replication effectivefor propagation in E. coli and other prokaryotic cells; (Greenfeder, S.A., et al., J. Biol. Chem. 270:13757 (1995)) an ampicillin resistancegene for selection of plasmid-containing prokaryotic cells; (Polan, M.L., et al., Am. J. Obstet. Gynecol. 170:1000 (1994)) an SV40 origin ofreplication for propagation in eukaryotic cells; (Carinci, Mora, M., etal., Prog. Clin. Biol. Res. 349:205 (1990)) a CMV promoter, apolylinker, an SV40 intron, and a polyadenylation signal arranged sothat a cDNA conveniently can be placed under expression control of theCMV promoter and operably linked to the SV40 intron and thepolyadenylation signal by means of restriction sites in the polylinker.

A DNA fragment encoding the entire T1R-like ligand II precursor and anHA tag fused in frame to its 3′ end is cloned into the polylinker regionof the vector so that recombinant protein expression is directed by theCMV promoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

Plasmid Construction

The T1R-like ligand II cDNA of the deposited clone is amplified usingprimers that contain convenient restriction sites, much as describedabove regarding the construction of expression vectors for expression ofT1R-like ligand II in E. coli. To facilitate detection, purification andcharacterization of the expressed T1R-like ligand II, one of the primerscontains a hemagglutinin tag (“HA tag”) as described above.

One of ordinary skill in the art will understand that the full-lengthT1R-like ligand II protein (amino acid about −26 to about 203 in SEQ IDNO:2) can be expressed in COS cells using suitable 5′ and 3′oligonucleotide primers.

The cDNA sequence encoding the extracellular domain of the full lengthT1R-like ligand II in the deposited clone is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene. The 5′ primer has the following sequence: 5′ CGC GGA TCC GCC ATCATG GGC GAC AAG ATC TGG 3′ (SEQ ID NO: 6), containing the underlinedBamH1 site and 18 nucleotides (nucleotides 55 to 72) of the T1R-likeligand II coding sequence set out in FIGS. 1A–B (SEQ ID NO:1).

The 3′ primer has the following sequence: 5′ CGC TCT AGA TCA AGC GTA GTCTGG GAC GTC GTA TGG GTA TCT ATC AAA GTT GCT TTC 3′ (SEQ ID NO:9),containing the underlined Xba I restriction site, a stop codon, an HAtag, and 18 nucleotides reverse and complementary to nucleotides 619–639of the TR1-like ligand II coding sequence set out in FIGS. 1A–B (SEQ IDNO:1).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamH I and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the T1R-like ligand II encodingfragment.

For expression of recombinant T1R-like ligand II, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989). Cells are incubated under conditions for expressionof T1R-like ligand II by the vector.

Expression of the T1R-like ligand II HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of T1R-like ligand protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No.37146]. Both plasmids contain the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357–1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107–143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64–68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pC4 contains for the expression of the gene of interest a strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular biology, March 1985, 438–4470)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521–530, 1985).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Pvull,and Nrul. Behind these cloning sites the plasmid contains translationalstop codons in all three reading frames followed by the 3′ intron andthe polyadenylation site of the rat preproinsulin gene. Other highlyefficient promoters can also be used for the expression, e.g., the humanβ-actin promoter, the SV40 early or late promoters or the long terminalrepeats from other retroviruses, e.g., HIV and HTLVI. For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g. G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding T1R-like ligand II protein is amplified usingPCR oligonucleotide primers specific to the amino terminal sequence ofthe T1R-like ligand II protein and to vector sequences 3′ to the gene.Additional nucleotides containing restriction sites to facilitatecloning are added to the 5′ and 3′ sequences respectively.

The cDNA sequence encoding the full length T1R-like ligand II in thedeposited clone is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene. The 5′ primer hasthe sequence 5′ CGC GGA TCC GCC ATC ATG GGC GAC AAG ATC TGG 3′ (SEQ IDNO: 6), containing the underlined BamH I restriction enzyme sitefollowed 18 nucleotides (nucleotides 55–72) of the sequence of T1R likeligand II in FIGS. 1A–B (SEQ ID NO:1). Inserted into an expressionvector, as described herein, the 5′ end of the amplified fragmentencoding T1R-like ligand II provides an efficient signal peptide. Anefficient signal for initiation of translation in eukaryotic cells, asdescribed by Kozak, M., J. Mol. Biol. 196: 947–950 (1987) isappropriately located in the vector portion of the construct.

The 3′ primer has the sequence 5′ GCG GGT ACC TCA CAA TGT TAC GTA CTCTAG 3′ (SEQ ID NO: 7), containing the underlined Asp 718 restrictionsite followed by a stop codon and 18 nucleotides reverse andcomplementary to nucleotides 754 to 771 of the T1R-like ligand II codingsequence in FIGS. 1A–B (SEQ ID NO:1). The restriction sites areconvenient to restriction enzyme sites in the CHO expression vectorPC-4.

The cDNA sequence encoding the extracellular domain of the full lengthT1R-like ligand II in the deposited clone is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene.

The 5′ primer has the sequence 5′ CGC GGA TCC GCC ATC ATG GGC GAC AAGATC TGG 3′ (SEQ ID NO:6) containing the underlined BamHI restrictionenzyme site and 18 nucleotides (nucleotides 55 to 72) of the T1R-likeligand II coding sequence in FIGS. 1A–B (SEQ ID NO:1). Inserted into anexpression vector, as described herein, the 5′ end of the amplifiedfragment encoding T1R-like ligand II provides an efficient signalpeptide. An efficient signal for initiation of translation in eukaryoticcells, as described by Kozak, M., J. Mol. Biol. 196: 947–950 (1987) isappropriately located in the vector portion of the construct.

The 3′ primer has the sequence 5′ CGC GGT ACC TCA TCT ATC AAA GTT GCTTTC 3′ (SEQ ID NO:8) containing the underlined Asp 718 restriction sitefollowed by a stop codon and 18 nucleotides reverse and complementary tonucleotides 619–636 of the T1R-like ligand II coding sequence set out inFIGS. 1A–B (SEQ ID NO:1).

The amplified T1R-like ligand II protein DNA are digested with BamHI andAsp 718. The vector pC4 is digested with BamHI and the digested DNAs arethen ligated together. The isolated fragment and the dephosphorylatedvector are then ligated with T4 DNA ligase. Insertion of the T1R likeligand II protein DNA into the BamH I restricted vector places the T1Rlike ligand II protein coding region downstream of and operably linkedto the vector's promoter. E. coli HB101 cells are then transformed andbacteria identified that contained the plasmid pC4 inserted in thecorrect orientation using the restriction enzyme BamHI. The ligationmixture is transformed into competent E. coli cells using standardprocedures as described, for example, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). The transformed culture isplated on ampicillin media plates which then are incubated to allowgrowth of ampicillin resistant colonies. Plasmid DNA is isolated fromresistant colonies and examined by restriction analysis and gel sizingfor the presence of the T1R-like ligand II-encoding fragment. Thesequence of the inserted gene is confirmed by DNA sequencing.

Example 3(c) Transfection of CHO-DHFR-Cells

Chinese hamster ovary cells lacking an active DHFR enzyme are used fortransfection. 5 μg of the expression plasmid C4 are cotransfected with0.5 μg of the plasmid pSVneo using the lipofecting method (Felgner etal., supra). The plasmid pSV2-neo contains a dominant selectable marker,the gene neo from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany)and cultivated from 10–14 days. After this period, single clones aretrypsinized and then seeded in 6-well petri dishes using differentconcentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (500 nM, 1 μM, 2 μtM, 5 μM). The same procedure isrepeated until clones grow at a concentration of 100 μM.

The expression of the desired gene product is analyzed by Western blotanalysis and SDS-PAGE. Expression of the T1R-like ligand II fusionprotein is detected by radiolabelling and immunoprecipitation, usingmethods described in, for example Harlow et al., Antibodies: ALaboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1988). To this end, two days after transfection,the cells are labeled by incubation in media containing ³⁵S-cysteine for8 hours. The cells and the media are collected, and the cells are washedand the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1%NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described byWilson et al. cited above. Proteins are precipitated from the celllysate and from the culture media using an HA-specific monoclonalantibody. The precipitated proteins then are analyzed by SDS-PAGE gelsand autoradiography. An expression product of the expected size is seenin the cell lysate, which is not seen in negative controls.

Example 4 Tissue Distribution of T1R-Like Ligand II Gene Expression

Northern blot analysis is carried out to examine expression levels ofthe T1R-like ligand II gene in human tissues, using methods describedby, among others, Sambrook et al., cited above. A cDNA probe containingthe entire T1R-like ligand II nucleotide sequence (SEQ ID NO:1) islabeled with ³²p using the rediprime™ DNA labelling system (AmershamLife Science), according to manufacturer's instructions. Afterlabelling, the probe is purified using a CHROMA SPIN-100™ column(Clontech Laboratories, Inc.), according to manufacturer's protocolnumber PT1200–1. The purified labelled probe is then used to examinevarious human tissues for expression of the T1R-like ligand II gene.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) and human immune system tissues (IM) are obtained from Clontech andare examined with labelled probe using ExpressHyb™ HybridizationSolution (Clontech) according to manufacturer's protocol numberPT1190–1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The disclosures of all patents, patent applications, and publicationsreferred to herein are hereby entirely incorporated by reference.

Example 5 Gene Therapy Using Endogenous T1R-Like Ligand II Gene

A method of gene therapy according to the present invention involvesoperably associating the endogenous T1R-like ligand II sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNumber WO 96/29411, published Sep. 26, 1996; International PublicationNumber WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932–8935 (1989); and Zijlstra et al., Nature342:435–438 (1989). This method involves the activation of a gene whichis present in the target cells, but which is not expressed in the cells,or is expressed at a lower level than desired. Polynucleotide constructsare made which contain a promoter and targeting sequences, which arehomologous to the 5′ non-coding sequence of endogenous T1R-like ligandII, flanking the promoter. The targeting sequence will be sufficientlynear the 5′ end of T1R-like ligand II so the promoter will be operablylinked to the endogenous sequence upon homologous recombination. Thepromoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousT1R-like ligand II sequence. This results in the expression of T1R-likeligand II in the cell. Expression may be detected by immunologicalstaining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the T1R-like ligand II locus,plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII.The CMV promoter is amplified by PCR with an XbaI site on the 5′ end anda BamHI site on the 3′ end. Two T1R-like ligand II non-coding sequencesare amplified via PCR: one T1R-like ligand II non-coding sequence(T1R-like ligand II fragment 1) is amplified with a Hindlll site at the5′ end and an Xba site at the 3′ end; the other T1R-like ligand IInon-coding sequence (T1R-like ligand II fragment 2) is amplified with aBamHI site at the 5′ end and a HindIII site at the 3′ end. The CMVpromoter and T1R-like ligand II fragments are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; T1R-like ligand IIfragment 1—XbaI; T1R-like ligand II fragment 2—BamHI) and ligatedtogether. The resulting ligation product is digested with HindIII, andligated with the HindIII-digested pUC 18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250–300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14–20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16–24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 6 Protein Fusions of T1R-Like Ligand II

T1R-like ligand II polypeptides of the invention are optionally fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of T1R-like ligand II polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See EP A 394,827; Traunecker, et al., Nature331:84–86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albuminincreases the halflife time in vivo. Nuclear localization signals fusedto T1R-Like Ligand II polypeptides can target the protein to a specificsubcellular localization, while covalent heterodimer or homodimers canincrease or decrease the activity of a fusion protein. Fusion proteinscan also create chimeric molecules having more than one function.Finally, fusion proteins can increase solubility and/or stability of thefused protein compared to the non-fused protein. All of the types offusion proteins described above can be made using techniques known inthe art or by using or routinely modifying the following protocol, whichoutlines the fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedherein. These primers also preferably contain convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector.

For example, if the pC4 (Accession No. 209646) expression vector isused, the human Fc portion can be ligated into the BamHI cloning site.Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and T1R-like ligand II polynucleotide, isolated by the PCRprotocol described in Example 1, is ligated into this BamHI site. Notethat the polynucleotide is cloned without a stop codon, otherwise afusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region:

-   GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGC    ACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC    ACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCC    ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA    ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA    GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA    GGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAA    GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTG    ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACA    TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC    CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC    AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC    TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCG    ACGGCCGCGACTCTAGAGGAT (SEQ ID NO:26)

Example 7 Isolation of Antibody Fragments Directed Against Polypeptidesof the Present Invention from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a large library of antibody fragments which contain reactivitiesagainst polypeptides of the present invention to which the donor may ormay not have been exposed (see e.g., U.S. Pat. No. 5,885,793incorporated herein in its entirety by reference).

Rescue of the Library:

A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO 92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harbouring the phagemid are used to inoculate50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin(2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five μml ofthis culture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU ofdelta gene 3 helper phage (M13 Δ gene III, see WO 92/01047) are addedand the culture incubated at 37° C. for 45 minutes without shaking andthen at 37° C. for 45 minutes with shaking. The culture is centrifugedat 4000 r.p.m. for 10 minutes and the pellet resuspended in 2 liters of2×TY containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and grownovernight. Phage are prepared as described in WO 92/01047.

M13 Δ gene III is prepared as follows: M13 Δ gene III helper phage doesnot encode gene III protein, hence the phage(mid) displaying antibodyfragments have a greater avidity of binding to antigen. Infectious M13 Δgene III particles are made by growing the helper phage in cellsharboring a pUC19 derivative supplying the wild type gene III proteinduring phage morphogenesis. The culture is incubated for 1 hour at 37°C. without shaking and then for a further hour at 37° C. with shaking.Cells are pelleted (IEC-Centra 8,4000 revs/min for 10 min), resuspendedin 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μgkanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phageparticles are purified and concentrated from the culture medium by twoPEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS andpassed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning of the library:

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100mg/ml or 10 mg/ml of a polypeptide of the present invention. Tubes areblocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 timesin PBS. Approximately 10¹³ TU of phage are applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used toinfect 10 ml of mid-log E. coli TG1 by incubating eluted phage withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 μg/ml ampicillin. The resultingbacterial library is then rescued with delta gene 3 helper phage asdescribed above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders:

Eluted phage from the 3rd and 4th rounds of selection are used to infectE. coli HB 2151 and soluble scfv is produced (Marks, et al., 1991) fromsingle colonies for assay. ELISAs are performed with microtitre platescoated with either 10 pg/ml of the polypeptide of the present inventionin 50 mM bicarbonate pH 9.6. Clones positive in ELISA are furthercharacterized by PCR fingerprinting (see e.g., WO 92/01047) and then bysequencing.

Example 8 Production of an Antibody

Hybridoma Technology:

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing T1R-like ligand II polypeptide(s) areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of T1R-likeligand II polypeptide(s) is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

Monoclonal antibodies specific for T1R-like ligand II polypeptide(s) areprepared using hybridoma technology. (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563–681 (1981)).In general, an animal (preferably a mouse) is immunized with T1R-likeligand II polypeptide(s) or, more preferably, with a secreted T1R-likeligand II polypeptide-expressing cell. Such polypeptide-expressing cellsare cultured in any suitable tissue culture medium, preferably inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225–232 (1981)). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the T1R-like ligand IIpolypeptide(s).

Alternatively, additional antibodies capable of binding to T1R-likeligand II polypeptide(s) can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theT1R-like ligand II protein-specific antibody can be blocked by T1R-likeligand II polypeptide(s). Such antibodies comprise anti-idiotypicantibodies to the T1R-like ligand II protein-specific antibody and areused to immunize an animal to induce formation of further T1R-likeligand II protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed herein. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Example 9 Method of Determining Alterations in the T1R-Like Ligand IIGene

RNA is isolated from entire families or individual patients presentingwith a phenotype of interest (such as a disease). cDNA is then generatedfrom these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95° C. for 30 seconds; 60–120 secondsat 52–58° C.; and 60–120 seconds at 70° C., using buffer solutionsdescribed in Sidransky, D., et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofT1R-Like Ligand II are also determined and genomic PCR products analyzedto confirm the results. PCR products harboring suspected mutations inT1R-Like Ligand II is then cloned and sequenced to validate the resultsof the direct sequencing.

PCR products of T1R-Like Ligand II are cloned into T-tailed vectors asdescribed in Holton, T. A. and Graham, M. W., Nucleic Acids Research,19:1156 (1991) and sequenced with T7 polymerase (United StatesBiochemical). Affected individuals are identified by mutations inT1R-Like Ligand II not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in the T1R-Like Ligand II gene. Genomic clones isolatedusing techniques known in the art are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson, Cg. et al., Methods Cell Biol.35:73–99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to theT1R-Like Ligand II genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of T1R-Like Ligand II (hybridized bythe probe) are identified as insertions, deletions, and translocations.These T1R-Like Ligand II alterations are used as a diagnostic marker foran associated disease.

Example 10 Method of Detecting Abnormal Levels of T1R-Like Ligand II ina Biological Sample

T1R-Like Ligand II polypeptides can be detected in a biological sample,and if an increased or decreased level of T1R-Like Ligand II isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect T1R-Like LigandII in a sample, preferably a biological sample. Wells of a microtiterplate are coated with specific antibodies to T1R-Like Ligand II at afinal concentration of 0.2 to 10 μg/ml. The antibodies are eithermonoclonal or polyclonal and are produced using technique known in theart. The wells are blocked so that non-specific binding of T1R-LikeLigand II to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining T1R-Like Ligand II. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedT1R-Like Ligand II.

Next, 50 μl of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25–400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate(NPP) substrate solution is then added to each well and incubated 1 hourat room temperature to allow cleavage of the substrate and flourescence.The flourescence is measured by a microtiter plate reader. A standardcurve is preparded using the experimental results from serial dilutionsof a control sample with the sample concentration plotted on the X-axis(log scale) and fluorescence or absorbance on the Y-axis (linear scale).The T1R-Like Ligand II polypeptide concentration in a sample is theninterpolated using the standard curve based on the measured flourescenceof that sample.

Example 11 Method of Treating Increased Levels of T1R-Like Ligand IIUsing an Antagonist

The present invention relates to a method for treating an individual inneed of a decreased level of T1R-Like Ligand II biological activity inthe body comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of T1R-Like Ligand IIantagonist. Preferred antagonists for use in the present invention areT1R-Like Ligand II specific antibodies and antisense polynucleotides.

Antisense technology is used to inhibit production of T1R-Like LigandII. This technology is one example of a method of decreasing levels ofT1R-Like Ligand II polypeptide, preferably a soluble and/or secretedform, due to a variety of etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels ofT1R-Like Ligand II is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the isdetermined to be well tolerated.

In another example, a patient with increased levels of T1R-Like LigandII polypeptide receives a daily dose 0.1–100 μg/kg of an antagonist forsix consecutive days. Preferably, the antagonist is in a soluble and/orsecreted form.

Example 12 Method of Treating Decreased Levels of T1R-Like Ligand II

The present invention also relates to a method for treating anindividual in need of an increased level of T1R-Like Ligand IIbiological activity in the body comprising administering to such anindividual a composition comprising a therapeutically effective amountof T1R-Like Ligand II or an agonist thereof.

For example, a patient with decreased levels of T1R-Like Ligand IIpolypeptide receives a daily dose 0.1–100 μg/kg of agonist and/orpolypeptide for six consecutive days. Preferably, the agonist and/orpolypeptide is in a soluble and/or secreted form.

Example 13 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing soluble and/or mature T1R-Like Ligand II polypeptides, onto apatient. Generally, fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in tissue-culture medium andseparated into small pieces. Small chunks of the tissue are placed on awet surface of a tissue culture flask, approximately ten pieces areplaced in each flask. The flask is turned upside down, closed tight andleft at room temperature over night. After 24 hours at room temperature,the flask is inverted and the chunks of tissue remain fixed to thebottom of the flask and fresh media (e.g., Ham's F12 media, with 10%FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37° C. for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219–25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding T1R-Like Ligand II can be amplified using PCR primerswhich correspond to the 5′ and 3′ end encoding sequences respectively.Preferably, the 5′ primer contains an EcoRI site and the 3′ primerincludes a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is then used to transform E. coli HB101, which are then plated onto agar containing kanamycin for thepurpose of confirming that the vector contains properly insertedT1R-Like Ligand II.

The amphotropic pA317 or GP+am 12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the T1R-Like Ligand II gene is then added to the media andthe packaging cells transduced with the vector. The packaging cells nowproduce infectious viral particles containing the T1R-Like Ligand IIgene(the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether T1R-LikeLigand II protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 14 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) T1R-like ligand II sequences into an animal toincrease or decrease the expression of the T1R-like ligand IIpolypeptide. The T1R-like ligand II polynucleotide may be operativelylinked to a promoter or any other genetic elements necessary for theexpression of the T1R-like ligand II polypeptide by the target tissue.Such gene therapy and delivery techniques and methods are known in theart, see, for example, WO 90/11092, WO 98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; TabataH. et al., Cardiovasc. Res.35:470–479(1997); Chao J. et al., Pharmacol. Res. 35:517–522(1997);Wolff J. A. Neuromuscul. Disord. 7:314–318(1997); Schwartz B. et al.,Gene Ther. 3:405–411(1996); Tsurumi Y. et al., Circulation 94:3281–3290(1996) (incorporated herein by reference).

The T1R-like ligand II polynucleotide constructs maybe delivered byanymethod that delivers injectable materials to the cells of an animal,such as, injection into the interstitial space of tissues (heart,muscle, skin, lung, liver, intestine and the like). The T1R-like ligandII polynucleotide constructs can be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the T1R-like ligand II polynucleotides may also bedelivered in liposome formulations (such as those taught in Felgner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126–139 and Abdallah B. et al.(1995) Biol. Cell 85(1):1–7) which can be prepared by methods well knownto those skilled in the art.

The T1R-like ligand II polynucleotide vector constructs used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Any strong promoter known to those skilled in the art canbe used for driving the expression of DNA. Unlike other gene therapiestechniques, one major advantage of introducing naked nucleic acidsequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The T1R-like ligand II polynucleotide construct can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed herein.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked T1R-like ligand II polynucleotide injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05 g/kgbody weight to about 50 mg/kg body weight. Preferably the dosage will befrom about 0.005 mg/kg to about 20 mg/kg and more preferably from about0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skillwill appreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked T1R-likeligand II polynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected T1R-like ligand II polynucleotidein muscle in vivo is determined as follows. Suitable T1R-like ligand IItemplate DNA for production of mRNA coding for T1R-Like Ligand IIpolypeptide is prepared in accordance with a standard recombinant DNAmethodology. The template DNA, which may be either circular or linear,is either used as naked DNA or complexed with liposomes. The quadricepsmuscles of mice are then injected with various amounts of the templateDNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The T1R-like ligand II template DNA is injected in 0.1 ml ofcarrier in a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 μmcross-section of the individual quadriceps muscles is histochemicallystained for T1R-like ligand II protein expression. A time course forT1R-like ligand II protein expression may be done in a similar fashionexcept that quadriceps from different mice are harvested at differenttimes. Persistence of T1R-like ligand II DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using T1R-like ligand II naked DNA.

Example 15 Bioassay for the Effect of T1R-Like Ligand II onHematopoietic Progenitor Cells and/or Differentiation

Mouse bone marrow cells are used as target cells to examine the effectof T1R-like ligand II polypeptides of the invention on hematopoieticprogenitor cells and/or differentiation. Briefly, unfractionated bonemarrow cells are first washed 2× with a serum-free IMDM that issupplemented with 10% (V/V) BIT (Bovine serum albumin, Insulin andTransferrin supplement from Stem Cell Technologies, Vancouver, Canada).The washed cells are then resuspended in the same growth medium andplated in the 96-well tissue culture plate (5×104 cells/well) in 0.2 mlof the above medium in the presence or absence of cytokines and T1R-likeligand II. Stem cell factor (SCF) and IL-3 are included as positivemediators of cell proliferation. Cells are allowed to grow in a lowoxygen environment (5% CO₂, 7% O₂, and 88% N₂) tissue culture incubatorfor 6 days. On the sixth day, 0.5 μCi of Tritiated thymidine is added toeach well and incubation is continued for an additional 16–18 hours, atwhich point the cells are harvested. The level of radioactivityincorporated into cellular DNA is determined by scintillationspectrometry and reflects the amount of cell proliferation.

The studies described in this example test the activity of T1R-likeligand II polypeptides of the invention. However, one skilled in the artcould easily modify the exemplified studies to test the activity ofT1R-like ligand II polynucleotides (e.g., gene therapy), agonists,and/or antagonists of T1R-like Ligand II. Potential agonists would beexpected to inhibit hematopoietic cell proliferation in the presence ofSCF and/or IL3 and/or to increase the inhibition of cell proliferationin the presence of cytokines and T1R-like ligand II in this assay.Potential antagonists would be expected to reduce the inhibition of cellproliferation in the presence of cytokines and T1R-like ligand II inthis assay.

Example 16 Bioassay for the Effect of T1R-Like Ligand II on IL-3 and SCFStimulated Proliferation and Differentiation of Hematopoietic ProgenitorCells

To determine if T1R-like ligand II polypeptides of the invention inhibitspecific hematopoietic lineages, mouse bone marrow cells are firstwashed 2× with a serum-free IMDM that is supplemented with 10% (V/V) BIT(Bovine serum albumin, Insulin and Transferrin supplement from Stem CellTechnologies, Vancouver, Canada). The washed cells are then resuspendedin the same growth medium and plated in the 96-well tissue culture plate(5×104 cells/well) in 0.2 ml of the above medium in the presence of IL-3(1 ng/ml) plus SCF (5 ng/ml) with or without T1R-like ligand II. Cellsare allowed to grow in a low oxygen environment (5% CO₂, 7% O₂, and 88%N₂) tissue culture incubator, and after 7 days, analyzed for expressionof differentiation antigens by staining with various monoclonalantibodies and FACScan.

The studies described in this example test the activity of T1R-likeligand II polypeptides of the invention. However, one skilled in the artcould easily modify the exemplified studies to test the activity ofT1R-like ligand II polynucleotides (e.g., gene therapy), agonists,and/or antagonists of T1R-like Ligand II. Potential agonists tested inthis assay would be expected to inhibit cell proliferation in thepresence of cytokines and/or to increase the inhibition of cellproliferation in the presence of cytokines and T1R-like ligand II.Potential antagonists tested in this assay would be expected to reducethe inhibition of cell proliferation in the presence of cytokines andT1R-like ligand II.

Example 17 Effect of T1R-Like Ligand II on IL-3 and SCF StimulatedProliferation and Differentiation of Lin-Population of Bone Marrow Cells

A population of mouse bone marrow cells enriched in primitivehematopoietic progenitors can be obtained using a negative selectionprocedure, where the committed cells of most of the lineages are removedusing a panel of monoclonal antibodies (anti cd1 1b, CD4, CD8, CD45R andGr-1 antigens) and magnetic beads. The resulting population of cells(lineage depleted cells) are plated (5×104 cells/ml) in the presence orabsence of T1R-like ligand II polypeptide of the invention (in a rangeof concentrations) in a growth medium supplemented with IL-3 (5 ng/ml)plus SCF (100 ng/ml). After seven days of incubation at 37° C. in ahumidified incubator (5% CO₂, 7% O₂, and 88% N₂ environment), cells areharvested and assayed for the HPP-CFC, and immature progenitors. Inaddition, cells are analyzed for the expression of certaindifferentiation antigens by FACScan. Colony data is expressed as meannumber of colonies +/−SD) and are obtained from assays performed in sixdishes for each population of cells.

Example 18 T1R-Like Ligand II Stimulates the Proliferation of BoneMarrow CD34+ Cells

This assay was based on the ability of human CD34+ to proliferate inpresence hematopoietic growth factors and evaluated the ability ofisolated T1R-like ligand II polypeptides expressed in mammalian cells tostimulate proliferation of CD34+ cells.

It has been previously shown that only most mature precursors willrespond to a single signal. More immature precursors require at leasttwo signals to respond. Therefore, to test the effect of T1receptor-like ligand II polypeptides on hematopoietic activity of a widerange of progenitor cells, the assay contained T1R-like ligand II inpresence or absence of other hematopoietic growth factors. Isolatedcells were cultured for 5 days in the presence of Stem Cell Factor (SCF)in combination with tested supernatant. SCF alone has a very limitedeffect on the proliferation of bone marrow (BM) cells, acting in suchconditions only as a “survival” factor. However, combined with anyfactor exhibiting stimulatory effect on these cells (i.e. IL-3), SCFwill cause a synergistic effect. Therefore, if the tested polypeptidehas a stimulatory effect on hematopoietic progenitors, such activity canbe easily detected. Since normal BM cells have a low level of cyclingcells, it is likely that any inhibitory effect of the polypeptides ofthe invention, or agonists or antagonists thereof, might not bedetected, accordingly, assays for an inhibitory effect on progenitors ispreferably tested in cells that are first subjected to in vitrostimulation with SCF+IL+3, and then contacted with the compound that isbeing evaluated for inhibition of such induced proliferation.

Briefly, CD34+ cells were isolated using methods known in the art. Thecells were thawed and resuspended in medium (QBSF 60 serum-free mediumwith L-glutamine (500 ml) Quality Biological, inc. Gaithersburg, Md.,Cat# 160–204–101). After several gentle centrifugation steps at 200×g,they were allowed to rest for one hour. The cell count was then adjustedto 2.5×10⁵ cells/ml. During this time, 100 μl of sterile water was addedto the peripheral wells of a 96-well plate. The cytokines that weretested with T1 receptor-like ligand II in this assay were rhSCF (R&DSystems, Minneapolis, Minn., Cat# 255-SC) at 50 ng/ml alone and incombination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μlSID (supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells wereadded to the media which was already present in the wells to allow for afinal total volume of 100 μl. The plates were then placed in a 37° C./5%CO₂ incubator for five days.

Eighteen hours before the assay was harvested, 0.5 μCi/well of [3H]Thymidine was added in a 10 μl volume to each well to determine theproliferation rate. The experiment was terminated by harvesting thecells from each 96 well plate to a filtermat using the Tomtec Harvester96. After harvesting, the filtermats were dried, trimmed and placed intoOmniFilter assemblies consisting of one OmniFilter plate and oneOmniFilter Tray. 60 μl Microscint was added to each well and the platesealed with TopSeal-A press-on sealing film. A bar code 15 sticker wasaffixed to the first plate for counting The sealed plates were thenloaded and the level of radioactivity determined via the Packard TopCount and the printed data collected for analysis. The level ofradioactivity reflected the amount of cell proliferation.

FIG. 4 shows the results of one such assay using isolated T1R-likeligand II polypeptides expressed in mammalian cells. The values wereaveraged and standard deviations calculated using Microsoft 98 Excel.Hits are determined by averaging all of the mean values for the SID Litesupernatants and controls and adding 1 SD to that value. Any SID Litesupernatants whose average value exceeds this value are considered ashits.

The studies described in the above example test the activity of T1R-likeligand II polypeptides of the invention. However, one skilled in the artcould easily modify the exemplified studies to test the activity ofT1R-like ligand II polynucleotides (e.g., gene therapy), antibodies,agonists, and/or antagonists and fragments and variants thereof. As anonlimiting example, potential antagonists tested in this assay would beexpected to inhibit cell proliferation in the presence of cytokinesand/or to increase the inhibition of cell proliferation in the presenceof cytokines and T1R-like ligand II. Potential agonists tested in thisassay would be expected to reduce the inhibition of cell proliferationin the presence of cytokines and T1R-like ligand II.

1. A method of inhibiting bone marrow cell proliferation comprisingadministering an antibody that specifically binds to an amino acidsequence selected from the group consisting of: a) amino acids 1–168 ofSEQ ID NO:2; and b) amino acids 1–203 of SEQ ID NO:2; wherein theproliferation of acid bone marrow cells is inhibited.
 2. The method ofclaim 1, wherein said administration is in vitro.
 3. The method of claim1, wherein said administration is in vivo.
 4. The method of claim 1,wherein said antibody specifically binds to amino the amino acidsequence of (a).
 5. The method of claim 1, wherein said antibodyspecifically binds to the amino acid sequence of (b).
 6. The method ofclaim 1, wherein the antibody is a monoclonal antibody.
 7. The method ofclaim 1, wherein the antibody is a human antibody.
 8. The method ofclaim 1, wherein the antibody is selected from the group consisting of:(a) a polyclonal antibody; (b) a chimeric antibody; (c) a humanizedantibody; (d) a single chain antibody; and (e) a Fab fragment.
 9. Themethod of claim 1, wherein the antibody is recombinantly produced. 10.The method of claim 1, wherein the antibody is labeled.